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Nowadays, soil contamination by total petroleum hydrocarbons is still one of the most widespread forms of contamination. Intervention technologies are consolidated; however, full-scale interventions turn out to be not sustainable. Sustainability is essential not only in terms of costs, but also in terms of restoration of the soil resilience. Bioremediation has the possibility to fill the gap of sustainability with proper knowledge. Bioremediation should be optimized by the exploitation of the recent “omic” approaches to the study of hydrocarburoclastic microbiomes. To reach the goal, an extensive and deep knowledge in the study of bacterial and fungal degradative pathways, their interactions within microbiomes and of microbiomes with the soil matrix has to be gained.
- culturable and unculturable microbes
- Bioremediation
- Soil
1. The Soil Scenario
Soil crude oil and total petroleum hydrocarbon (TPH) contaminations are mainly consisting of weathered ones, except for fresh accidental spills. Indeed, processes of adsorption, photo transformation, biological transformation and volatilization of the diverse crude oil and TPH components are at the base of the transformation of fresh contamination in weathered ones in soil [7]. The process of weathering chemical structures in the environment is strictly dependent on the complexity of their chemical structures, which determine potential volatilization, water solubility, and loss of bioavailability [8]. In soil, the weathering of TPH is principally consistent with their sorption to the soil organic matter (SOM). The SOM in terms of amount [9] and nature [10] is mainly responsible for the decrease of bioavailability of TPH in soil. In fact, partitioning processes of TPH in the SOM entrap the contamination in soil micropores [11,12]. Once the contamination is entrapped in the SOM, the establishment of diverse processes of interaction among chemical structures with similar moieties (e.g., humic fraction of the SOM and organic contaminants with aromatic moieties) occurs. These interactions consist in dipole–dipole, dipole-induced dipole and hydrogen bonding [13]. Moreover, interactions between TPH and the soil mineral constituents also occur [14,15,16], strengthening the decrease in TPH bioavailability in soil, especially with the aging of the contamination. The higher is the molecular weight of the organic contaminants, the higher the number of aromatic moieties and functional groups capable to interact with the SOM. Consequently, a weathered oil-contaminated soil is usually dominated by high molecular weight hydrocarbons, significantly recalcitrant to biodegradation [17,18]. In this context, the high molecular weight hydrocarbons are actually less toxic than the low molecular one, since these latter ones are less bioavailable for plants and microorganisms. The lag phase of bacterial growth was described as longer in the presence of higher concentrations of bioavailable hydrocarbons, and plant growth is inhibited by their presence since their hydrophobicity decreases the soil capacity to absorb water and nutrients [7].
On the other hand, it is generally accepted that microbes can eventually transform all the surrounding organic material over time. In particular, contaminants in environmental matrices exert a selective pressure on microbial communities. Speciation processes eventually occur and with time microbes, develop strategies to transform contaminants. Historically contaminated matrices are a useful reservoir of these organisms. Microbial strategies for contaminant transformation include not only degradative pathways but also the increment in their bioavailability and even their immobilization to contrast their toxicity (e.g., polycondensation or humification of the organic matter). Consequently, it is not surprising that the persistence of the TPH fractions in the soil is associated with the speciation of microbial strains that develop the capacity to transform the fraction with time [19].
Microbial biodegradation and biotransformation of organics vary in accordance with their chemical structures and the functional capabilities of the microbial community, specialized for the transformation of the parent molecule and of its intermediates of degradation. These latter ones are actually significantly important for the fate of pollutants in the environment, since in some cases, degradation products of the primary pollutants are more prone to subsequent transformations than the parent contaminant and, therefore, may be mineralized [20]. Other degradation products, such as polyaromatic chemical structures, are more susceptible to interaction with the SOM [21]. At the same time, the microbial catabolism is driven by pollutant concentration, since the activation of the microbial-degrading metabolisms needs a minimum threshold of pollutant concentration [22]. To reach these threshold levels, the bioavailability or the water solubility of the contaminant is mandatory. Consequently, in soil, TPH biodegradation rates are based also on the sorbed phase of the contamination [23,24,25,26,27]. Indeed, microorganisms reach the sorbed phase of the contamination because of their capacity to produce surfactants, responsible for both the increase in contaminant bioavailability and for facilitating the fixation of microorganisms onto the surface of the sorbed phase contaminants [28,29]. Thus, it is evident that there are a plethora of chemical-physical and biological elements that determine the destiny of organic pollutants in soil, and the condition is complicated by the complexity of the composition of the contamination.
3. Hydrocarburoclastic Bacterial Pathways
The aerobic bacterial metabolic pathways involved in TPH transformation are mainly intracellular. Regarding the aromatic fraction of TPH, the mechanisms developed by bacterial cells to assimilate aromatic compounds have been fixed and optimized by natural selection, giving rise to functionally catabolic pathways organized in a funnel-like topology. In fact, a wide diversity of aromatics is directed via different peripheral pathways to a few key central intermediates. The latter are subjected to de-aromatization and further conversion to intermediary metabolites, such as acetyl-CoA, succinyl-CoA or pyruvate, via central pathways [30]. Hydroxylating oxygenases and ring-cleavage dioxygenases are responsible, respectively, for the hydroxylation and oxygenolytic cleavage of the aromatic rings. The products of these oxidative passages converge both on cathecolic structures that are subjected to ortho or meta cleavage by intradiol or extradiol (type I and II) dioxygenases, respectively, and on non-catecholic structures such as gentisate, homogentisate, monohydroxylated aromatic acids and heteroaromatic flavonols, that are subject of ring cleavage by dedicated type III extradiol dioxygenases [31]. Moreover, dioxygenases such as the CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) are involved in the transformation of N-heteroaromatic compounds [32].
Regarding the TPH saturated fraction, this is composed by n-alkane whose oxidation is intracellular and initiated by oxygenases that introduce oxygen atoms into n-alkanes by four different pathways. The terminal oxidation pathway [33] is involved in the oxidation of the n-alkanes terminal methyl group. The product of the reaction is a primary alcohol, further oxidized by alcohol dehydrogenases and aldehyde dehydrogenases to fatty acid that enters in β-oxidation [34]. The termini of the n-alkane can be oxidated to the corresponding fatty acid without breaking the carbon chain by the biterminal oxidation pathway. The product of the reaction is a ω-hydroxy fatty acid, further converted to a dicarboxylic acid, entering in β-oxidation [34,35,36]. Subterminal oxidation has been also observed to form primary alcohols and secondary alcohols or methyl acetone with the same chain length as the substrate [37]. In Acinetobacter sp. strain HO1-N [38], the n-alkanes are oxidized to form n-alkyl hydroperoxides and then peroxy acids, alkyl aldehydes and finally, fatty acids. A dioxygenase is responsible for the first step of oxidation [39].
The water solubility and consequent bioavailability of both the aromatic and saturated fraction of TPH are low. The latter is eventually increased by the capability of most of the hydrocarburoclastic bacteria to produce biosurfactants. Biosurfactants are of diverse chemical compositions such as glycolipids, fatty acids, lipopeptides and lipoproteins, phospholipids and neutral lipids [40]. Biosurfactants are amphiphilic molecules with alkyl chains linked to sugar molecules, resulting in hydrophobic and hydrophilic regions, respectively [41], reducing the surface tension at the water/oil interface, leading to emulsification of hydrophobic moieties [42]. Indeed, biosurfactants enhance the bioavailability of contaminants for microbial degradation, by improving the solubilization of hydrocarbons in water layers, where bacterial metabolism and spreading occur [43]. Hydrocarburoclastic bacteria have been described as capable to produce biosurfactants in situ, which promote their survival in hydrophobic compound-dominated environments [44]. Different bacterial genera are described as capable to produce biosurfactants, among others, Pseudomonas, Bacillus, Acinetobacter, Alcaligenes, Rhodococcus and Corynebacterium spp. [40,45,46]. The exploitation of hydrocarburoclastic bacteria producing biosurfactants find application in the acceleration of the bioremediation of polluted soil and sediments [46,47,48], even though fragmented information are available on chemical and physical properties of biosurfactants produced by hydrocarburoclastic bacteria during the hydrocarbon degradation process [49].
4. Microbial Interactions
Synergisms between microorganisms in the environment might be explained by the generalization of the ecological concept of K-r strategy, which basically explains two different approaches of nutrition of “organisms” sharing the same niche. The r-strategy consists of high growth rates with high availability of carbon sources, while K-strategy consists of slow rates of growth and low availability of carbon sources. K-strategists are favored under nitrogen limitation since K-strategists are able to decompose the SOM, recalcitrant to biodegradation, for mineral nitrogen acquisition [78,79]. In parallel, r-strategists commensally utilize the SOM-derived compounds solubilized by the K-strategists. The r-strategists are favored in presence of available carbon and higher nitrogen availability [80]. A significant shift in microbial community structure from K-strategists to r-strategists is observed with the increased availability of nitrogen and carbon [81]. The shift might be observed also in terms of fungal abundances (K-strategists) in favor of bacterial ones (r-strategists), in consequence of the capacity of fungi to transform the SOM. It might be possible that the real situation is more complicated but it is evident that fungi and bacteria share microhabitats, assembled into dynamic co-evolving communities, described in almost every ecosystem [82]. These communities include microbial species from a wide diversity of fungal and bacterial families [83]. Microbial interactions contribute to soil functions as well as, and eventually even more than, species diversity [84]. Generally, the co-occurrence and synergism between fungi and bacteria in the soil might be based on the bacterial capacity to utilize fungal-secreted metabolites and overcome fungal defense mechanisms [85]. On the other hand, an example of the synergisms between the two kingdoms is the one of fungal lignocellulose decomposers, Clitocybe and Mycena spp., that interact with potential N2-fixing bacteria, responsible for nitrogen deposition in soil during the decay of leaves [86,87]. Bacteria may contribute to nitrogen nutrition of fungi while fungi produce carbon sources for bacteria. Moreover, in oligotrophic habitat, fungi promote bacterial growth by nutrient and water transfer from fungal hyphae to the bacterial cells [50]. Model simulations show that fungal hyphae are spreading bacteria towards a source of contamination in environments where the active movement of bacteria towards pollutant reservoirs is limited by physical barriers [88,89,90]. In the context of crude oil bioremediation, it is worth mentioning that fungal hyphae have been described to mobilize PAHs by entrapping and transporting the latter in cytoplasmic vesicles [91], providing entrapped PAHs to hydrocarburoclastic bacteria [92]. In laboratory models of water-unsaturated environments, fungi transport the contamination from the water-depleted niches to the less-dried ones, where bacteria are blooming [93]. Moreover, fungi excrete organic molecules such as organic acids or polyols that activate bacterial chemotaxis towards the hyphae [94,95]. Indeed, fungi promote ecosystem functioning in heterogeneous habitats by transporting resources from high nutrient and water levels to nutrient-poor and dry areas.
Other than chemotaxis between fungi and bacteria, another useful chemical signal tool in the ecology is quorum sensing (QS). Quorum sensing enables bacteria to communicate by exchanging signaling molecules also referred to as “autoinducers”. Two types of autoinducers are described: intraspecific signals, which are used by the same organism within a population and interspecific signals, which are used for communication among the entire microbial community [96]. A strong correlation between the expression of genes involved in hydrocarbon depletion and quorum sensing has been observed; as an example, a positive correlation between quorum sensing and the expression of the 2,3-catechol dioxygenase of the meta-cleavage (lower) pathway for hydrocarbon degradation has been reported [97]. On the other hand, the same correlation has been observed for quorum sensing modulation of the level of transcription of dioxygenase genes in the upper BTEX oxidation pathway [98]. QS is involved also in fungal morphogenesis, fungal biofilm development, apoptosis and eventually, pathogenicity [99]. Moreover, QS has been described as involved in interkingdom signaling in mixed fungal-bacterial biofilms [100,101,102,103].
5. Taxonomical and Functional Metagenomics for Bioremediation
The updated scenario of the microbial processes of TPH transformation in contaminated soils reveals levels of complexity that risk to associate the exploitation of bio-based processes to uncertainty. In order to transform the bio-based approaches to soil decontamination in robust technologies, a comprehensive understanding of the physiology, ecology and phylogeny of the colonizing microbial consortia is mandatory. The capacity to isolate microbial specialists for the biodegradation of TPH and crude oils is crucial for evaluating the biodegradation process they catalyze, as well as the regulatory mechanisms that influence their activities in contaminated soils. Moreover, the sequencing of their genomes is mandatory to deposit sequences of interest for in silico analysis of all the soil matrices contaminated by the same class of contaminants. Nowadays, the application of “omics” techniques in the investigation of pure cultures gives the opportunity to assess the genetic diversity of specialist species and of all environmental microbes, to investigate genes responsible for contaminant degradation and their regulation. In this context, it should be mentioned that “omics” investigation can be exploited for both isolates and environmental samples [116,117,118] and the adoption of metagenomics, transcriptomics and proteomics offers the opportunity to design new approaches to the management of processes dedicated to the restoration of the environment [119,120]. These techniques can be exploited to study the organization, interdependence, physiology, ecology and phylogeny of environmental microbiomes, comprising the hydrocarbuclastic ones. The study of microbiomes cannot be limited to culturomic approaches. Indeed, the large majority of microorganisms colonizing natural habitats are either uncultivable or extremely difficult to cultivate. Metagenomics is designed to study these microbes [121], with the recovering of DNA sequences from environmental DNA, offering the opportunity to discover genes and biodegradative pathways [122,123]. The correspondence between the kinetics of decontamination and the variations of the microbial ecology is mandatory to assess the microbial populations that are involved in the decontamination of TPH-contaminated soils [48]. The predictive functional metagenomics have been exploited to dissect bacterial functions that are dominant in processes of TPH depletion in soils, synergically catalyzed by fungi and saprophytic bacteria, introducing possible mechanisms of interaction between the fungal and the bacterial hydrocarburoclastic community in an aged TPH soil contamination [48].
The extremely useful functional metagenomic analysis can be successfully based also on the cloning of environmental DNA fragments in selected microbial hosts, successively screened for environmental functions of interest. The sequencing of the fragments cloned in the selected hosts leads to the sequencing of gene coding for these functions [124], enriching environmental databases on pollutant-degrading genomic sequences of known and unknown, isolated and not yet isolated, microbial candidates, facilitating in silico analysis of all the environmental matrices, affected by the same contamination. The described functional analysis combined with taxonomic metabarcoding and the derived predictive functional analysis should be considered as the bases for developing a robust predictive instrument to infer the functions that the microbial communities and colonizing contaminated matrices can express and, consequently, the functions to be exploited to complete and even accelerate a decontamination process. Due to the availability of a plethora of metagenomic, in silico functional and wet-lab functional data, bioinformatic processing applications for environmental DNA sequence-based screening of metagenomes are extremely important and they tend to be adopted among other methodological approaches to design bio-based technologies for the recovery of contaminated matrices. Among others, a list of the most important, frequently utilized and open-source pipelines of analysis follows.
Meta Genome Analyzer (MEGAN) analyzes large amounts of metagenomic sequence data [125] comparing metagenomic and metatranscriptomic data both functionally and taxonomically, mapping reads to the NCBI, SEED, COG and KEGG classificators. The tool is robust and user-friendly.
Community Cyberinfrastucture for Advanced Microbial Ecology Research and Analysis (CAMERA) is a database and computational tools providing the possibility for depositing, locating, analyzing and sharing data about microbial biology [126,127]. CAMERA offers the opportunity to deposit and work with metadata relevant to environmental metagenome datasets with annotations in a semantically aware environment. The user can use semantic queries to search in the database. CAMERA might be defined as a complete genome-analysis tool allowing users to analyze both metagenome and genome data.
Subsystem Technology for metagenomes (MG-RAST) is an automated platform providing quantitative insight into microbiomes based on their sequence data [128]. The pipeline performs similarity-based annotation on nucleic acid datasets, clustering and protein prediction, phylogenetic and metabolic reconstructions of genomes and metagenomes.
Phylogenetic metabarcoding is fundamental to study the microbial ecologies of any environmental niches. However, the molecular tool does not provide direct evidence of the community functional capabilities. PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), now PICRUSt2 [129], offers a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using phylogenetic information, PICRUSt2 infers key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty.
Indeed, metagenomics is fundamental to understanding the composition and the ecology of hydrocarburoclastic microbiota that can be defined as bucket-brigades for the degradation of TPH and crude oils in soils. Their functional analysis is fundamental to recover hydrocarburoclastic microbiomes, combining taxonomy and functional information, providing the instruments to deepen the information provided by a conceptual model of a contaminated site; in fact, the latter can comprise not only data about the stratigraphy, geological characteristics and chemical profiles of the contamination in the contaminated site. Indeed, the site will be characterized also in terms of the colonizing microbial communities, whose functional traits will be automatically inferred by the exploitation of data provided by the constantly increasing number of metagenomic studies. In this direction, it is worth mentioning that the predictive metagenomic profiling successfully described phylogenetic and functional compositions of diverse oil-polluted sites around the world, allowing the inferring of metagenomic features including taxonomical markers and functional modules, to be used as biomarkers for the effective distinction between diverse oil-polluted sites [130].
This entry is adapted from the peer-reviewed paper 10.3390/environments9040052
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