Removal of Hydrocarbons and Other Related Chemicals Via the Rhizosphere of Plants
Recalcitrant organic toxic chemicals have been accumulating for decades as a consequence of industrial activity. Concerns about environmental contamination have been rising in the last three decades and therefore the need for soil remediation has become a priority. Although physicochemical techniques are currently the most efficient methods being used to remove contaminants, they are very expensive and therefore impractical in many locations. The use of plants in the bioremediation of soils has been proposed as an attractive strategy; however, plants lack the extraordinary biodegradative capabilities of microorganisms. Consequently rhizoremediation, a technology which combines microorganisms that eliminate contaminants in the plant roots which provide nutrients for these microorganisms, has emerged. To design a successful rhizoremediation strategy, microorganisms need to proliferate in the root system, and the bacterial catabolic pathways have to be operative. Recent advances in these aspects, together with some techniques to improve biodegradation in the rhizosphere will be presented in this chapter.
Industrialization has allowed human development and higher living standards; however, these benefits have come at a high price in environmental terms. The inappropriate use of fertilizers and pesticides to improve crop production to cope with the needs of a growing population is leading to the contamination of underground water by nitrates and to the pollution of soil and surface water. The greater energy needs of society have increased CO2 production which is now one of the main concerns for governments. Industrial waste has resulted in the accumulation of recalcitrant organic chemicals such as DDT, lindane, triazines, and polychlorinated biphenyls (PCBs). In addition, soils are being gradually affected by the deposition of fuel particles enriched in polycyclic aromatic hydrocarbons (PAHs), as well as benzene and other chemicals derived from petrochemical activities. Recently, an increase in contaminants derived from personal care products and pharmaceuticals (PCPPs) has been detected in water. All these contaminants provoke an adverse effect on ecosystems and are a serious threat to human health. Due to their toxicity and in many cases their carcinogenic, teratogenic, endocrine disruption and mutagenic properties, a number of these chemicals have been declared pollutants and their removal is a priority for Environmental Protection Agencies. The removal of pollutants from air, water, and soil requires different technologies, and in this chapter, we will focus on the removal of contaminants from soil. Removal of pollutants from soil is a very complex and expensive procedure as it depends on numerous uncontrollable factors. Most of the successful remediation examples have included the physical removal of the contaminated soil (excavation, truck, and dump) and/or physical and/or chemical treatments; however, these techniques are quite expensive and involve the disruption of the soil structure (Gómez-Sagasti et al. 2016). In general, biological treatments are cheaper than physicochemical ones; however, they have been used in recent years with different degrees of success (Segura and Ramos 2013). The success of biological pollutant removal depends on the particular bioremediation strategy, the soil properties, age of the contamination, initial concentration of the contaminants, and obviously on the contaminant’s toxicity (dos Santos and Maranho 2018). On-site techniques such as land-farming or composting are promising options, but they involve the manipulation of soils and sometimes provoke the mobilization of the contaminant. In situ techniques, such as inoculation with microorganisms encoding the desired catalytic properties or bioaugmentation with native populations, are unreliable (Colleran 1997) and improving them by soil fertilization is expensive.
2 Phytoremediation Strategies
As an alternative to the failure of bioaugmentation, phytoremediation, the utilization of plants for the removal of pollutants, has been proposed as an attractive strategy to achieve the efficient elimination of pollutants.
The generic term, phytoremediation, is used to describe the elimination of contaminants by the plants themselves or in combination with root colonizing microorganisms, and it encompasses different strategies (Salt et al. 1998). In phytostabilization, plants either physically or by the action of the root exudates, help to sequester the contaminant to the soil particles thus making them less bioavailable (Burges et al. 2017); phytovolatilization is the term used when plants take up the contaminant from the soil and transform it into a volatile compound that is released into the atmosphere by transpiration for dispersal (Limmer and Burken 2016); phytoextraction is a technology used when plants are able to accumulate toxic compounds in the harvestable parts such as stems and leaves (Burges et al. 2017); rhizoremediation consists of the elimination of the contaminant by the microorganisms in the rhizosphere (Kuiper et al. 2004a); and phytoremediation as a strict term refers to the transformation of the contaminant by the metabolism of the plant (Macek et al. 2000).
Because plants are easy to monitor and agricultural technologies are constantly being developed, a priori the easiest way to design a phytoremediation protocol would be to use a plant as the single degradative organism. Many plants are able to transform (but in general not mineralize) some contaminants via their unique secondary metabolism, which is represented by the capacity of members of the cytochrome P450 super family of proteins to activate the contaminant that is subsequently conjugated with glutathione, malonyl, or glucosyl residues. The conjugated compound is then accumulated in vacuoles or in the plant cell wall (Burken 2004). These transformation steps are similar to those which take place in the liver of animals and led to the term “green liver” to designate the capacity of the plant to transform contaminants into less toxic compounds. Moreover, pollutants containing amino or hydroxyl groups can undergo rapid transformation by root-associated or extracellular enzymes (Shaw and Burns 2003).
Unfortunately plants do not possess the extraordinary repertoire of degradative enzymes of microorganisms, and therefore, the utilization of phytoremediation strategies based exclusively on plant activities cannot be used universally.
Due to their impressive degradation capacity for xenobiotic compounds, microorganisms have long been studied and assayed in bioremediation treatments. In contrast to the successful isolation of microorganisms with specific biodegradation properties, their reintroduction into soil to remove pollutants has been frequently unsuccessful. In some cases it has been reported that reintroduced microorganisms are unable to establish themselves in the soil. Although knowledge regarding the behavior of microorganisms in the environment is incomplete, it is thought that nutrient limitation, competition with indigenous microbiota, and/or limitations in their reactions under environmental stress conditions may account for the lack of success of (re)introduced microorganisms in contaminated soils. Some of these obstacles can be overcome by using rhizoremediation, a strategy that is based on the “rhizosphere effect” that relies on the plant excreting a number of chemicals that can be used as a carbon, nitrogen, sulfur, or phosphorous source by microorganisms to proliferate and reach high cell densities in the area surrounding the roots of the plant, i.e., the rhizosphere. The advantage of rhizoremediation is that plant roots provide a large surface on which microorganisms can proliferate by creating biofilms. Roots transport microorganisms through the soil both in terms of distribution and depth, and as mentioned above, the roots provide nutrients. Root penetration into the soil also facilitates oxygen exchange and thereby the proliferation of aerobic microorganisms and oxygen availability for the initial attack on many different chemicals in reactions mediated by mono- and dioxygenases.
3.1 Rhizoremediation Requirements
Rhizoremediation requires the augmentation of the desired bacteria (or consortia) in the laboratory to obtain a sufficient number of degradative bacteria to inoculate the seeds or roots of plants. After growing in the laboratory, the bacteria have to be able to proliferate on the root system under natural conditions (Fig. 1). Early experiments using in vivo expression technology (Rainey and Preston 2000; Ramos-González et al. 2005) and microarrays revealed the bacterial genes involved in the adaptation to the rhizosphere (Matilla et al. 2007; Attila et al. 2008). These findings indicated that after growth in the laboratory, bacteria have to reprogramme themselves to be able to live in the rhizosphere. Induction of genes related with changes in the transcriptional pattern of catabolic pathways to enable the utilization of the chemicals secreted by roots as carbon or nitrogen sources, and the induction of bacterial genes involved in oxidative stress are part of the response to the rhizosphere environment. It has also been repeatedly demonstrated that flagellar and chemotaxis genes are involved in root colonization (Lugtenberg et al. 2001; Scharf et al. 2016). These results revealed that the rhizosphere, although nutrient-rich compared with bulk soil, is a nutritionally challenging environment which is stressful to microorganisms due to the formation of reactive oxygen species produced by the respiration of root cells.
Identification of plant microbiomes (Bulgarelli et al. 2012) has confirmed that microbial populations in the rhizosphere depend on plant age, physiological status, and even the cultivar, supporting the idea that plant roots exert a certain selection toward the microorganisms living in their rhizosphere. The idea of specific plant-microbe communication is becoming more popular and, although most of the efforts in rhizoremediation have been focused on degradative bacteria, the utilization of different plants can also influence the outcome of contaminant removal. Microbial elimination of contaminants in the rhizosphere provides a positive effect for the plant as this leads to decreased pollutant concentrations in the area near the roots and thereby improved plant growth in contaminated areas (Rodriguez-Conde et al. 2016). Because of this mutual benefit, it has been proposed that plants can select for specific bacterial genotypes to be present on their roots. Experiments performed by Siciliano et al. (2002) demonstrated that the presence of the alkane monooxygenase gene was more prevalent in endophytic and rhizosphere microbial communities than in bulk soil contaminated with hydrocarbons. However, the results obtained when they studied the prevalence of xylene monooxygenase or naphthalene dioxygenase genes were just the opposite, their presence was higher in bulk soil microbial communities than those near to or on the plant. This effect was also dependent on the plant species used during the rhizoremediation strategy (Siciliano et al. 2003). Therefore, the combined influence of plants and contaminants determines the associated plant microbiota (Fig. 1). The presence of certain contaminants may affect the relative abundance of genotypes able to degrade this particular contaminant (Rodriguez-Conde et al. 2016) or the diversity of rhizosphere or endophytic bacteria (Liu et al. 2017). This has led to the hypothesis that the effectiveness of rhizoremediation strategies also depends on the selection of the best plant-bacteria combination for a specific contaminant (Thijs et al. 2016).
To complicate the general scheme, foreign bacteria also have to be able to compete with indigenous microorganisms suggesting another level of communication; they have to be able to sense their environment (Venturi and Keel 2016; Fig. 1). It is well known that bacteria are able to establish an intraspecific communication using auto-inducers that are synthesized when populations reach a critical density in a process called quorum sensing. The luxI gene encodes the auto-inducer synthase and the luxR codes for an auto-inducer-dependent activator responsible for coordinating the expression of several genes as well as the induction of the luxI gene (Stevens and Greenberg 1997; Fuqua and Greenberg 2002). luxR and luxI are generally located near to each other in the chromosome (Choudhary et al. 2013). However, as more microbial genomic information has become available in databases, it has been observed that many microorganisms encode several luxR-type genes that are not in the vicinity of any luxI and that there are more luxR genes than luxI. These genes have been named “orphan-luxR” or “luxR-solos” (Fuqua 2006). Some of these luxR-solos have been involved in intraspecific communication (microbe-microbe interactions) whilst others have been involved in inter-kingdom (plant-microbe) communication (Subramoni and Venturi 2009; Gonzalez and Venturi 2013).
Even if the reintroduced degradative-bacteria are: (i) able to grow with the nutrients available in the rhizosphere; (ii) able to cope with stressful environments; and (iii) able to compete with other microorganisms and to communicate with their environment, all these properties are useless if the degradative genes are not expressed in the rhizosphere. The rationale behind rhizoremediation strategies is that the rhizosphere environment contains additional C-sources that favor microbial growth which consequently accelerates degradation rates (Kuiper et al. 2004a; Lu et al. 2017). However, several reports have indicated that biodegradative genes may be affected by catabolic repression. Catabolic repression consists of the preferential utilization of some carbon sources over others; i.e., the toluene degradation pathway of the pWW0 plasmid is repressed in the presence of glucose or succinate (Duetz et al. 1996). Rentz et al. (2004) reported that root exudates from different plants inhibited the phenanthrene-degrading activity of Pseudomonas putida ATCC 17484, although these exudates allowed the growth of the strain. Other reports, however, indicate the fortuitous induction of catabolic genes in the rhizosphere (Casavant et al. 2003; Segura et al. 2017), probably as a consequence of the structural similarities between certain plant secondary metabolites and some xenobiotic chemicals (Singer et al. 2003).
3.2 Rhizoremediation Experiments
Different rhizoremediation strategies have been used to demonstrate the utility of this approach for the degradation of contaminants. Initially, bacterial strains that were well characterized for their capacity to degrade a certain contaminant were chosen, but frequently these strains were not well suited for survival in the rhizosphere. This has led to the current tendency to use rhizosphere bacteria. Using a double enrichment approach in which lindane degradation and root proliferation of bacteria were prerequisites for selection, Böltner et al. (2008) isolated Sphingomonas strains able to adhere to corn seeds at levels two orders of magnitude higher than the reference strain Sphingomonas UT26 and were able to colonize the root at approximately 5 × 108 CFUs (colony forming units) per gram of root. In controlled rhizoremediation experiments, these recently isolated strains were able to degrade 30% of the γ-hexaclorohexane of contaminated soil (0.5 mg per gram of soil) in 25 days. Amongst the disadvantages of this approximation are the lack of information about the degradative abilities of the bacteria and the lack of information about the ability of these bacteria to persist in the specific plant environment. These disadvantages can be alleviated if bacteria are isolated directly from the contaminated soil that has to be cleaned up (Zafra et al. 2017), but this approach requires additional experimentation in order to find the optimal bioaugmentation conditions to ensure that most of the new inoculum contains biodegradative bacteria.
Another approach is the utilization of bacteria that are generally present in contaminated environments. For example, different metagenomic experiments have revealed that in PAHs-contaminated soils, bacteria belonging to the genera Arthrobacter, Rhodobacter, Pseudomonas, Burkholderia, Bacillus, Mycobacterium, or to the Sphingomonadaecea family are frequently found to be the most abundant (Uhlik et al. 2012; Kappell et al. 2014; Rodriguez-Conde et al. 2016). Despite possible biases in the metagenomic analysis, it has been demonstrated that members of these genera and families are good PAH degraders (Lu et al. 2011). It is therefore interesting to test members of these families in rhizoremediation experiments. Utilization of single strains as well as consortia has demonstrated the utility of this approach (Zafra et al. 2017; Rodriguez-Conde et al. 2016).
Finally, the utilization of endophytes in the biodegradation of pollutants is an interesting option that is currently being explored (Afzal et al. 2014). In order to be successful, endophytes with interesting catabolic properties have to be isolated (Taghavi et al. 2009); alternatively, degradative bacteria in the rhizosphere have to be able to transfer interesting genetic determinants to plant endophytes (Mastretta et al. 2006). Natural horizontal transfer of the pTOM plasmid from Burkholderia cepacia isolated from yellow lupine, to poplar endophytes that then acquired the capacity to degrade toluene have already been demonstrated (Taghavi et al. 2005). For the successful utilization of this strategy, however, contaminants have to be transported into the plant interior and the transport mechanisms of the contaminants into the root cells have to be explored.
3.3 Monitoring Rhizoremediation
In rhizoremediation there are several parameters that have to be monitored: plant survival and growth, inoculum survival and contaminant elimination. Whilst initially, the number of colony forming units (CFUs) using selective media was almost the only method to follow up microbial survival and rhizosphere colonization, the advances in the utilization of reporter genes, such as the green-fluorescent protein (Gfp) and luciferase, have allowed the simplification of methods to monitor microbial survival in the rhizosphere (Ramos et al. 2000; Buddrus-Schiemann et al. 2010). However, these methods involve the genetic modification of the organisms and therefore, monitorization under natural conditions is mainly achieved by counting cells on selective media.
Contaminant biodegradation monitoring requires the extraction of the contaminant from soil and subsequent determination by analytical methods. These methods (mainly gas chromatography coupled to mass spectrometry [GC-MS] and high-pressure liquid chromatography [HPLC]) require expensive equipment and frequently, analyses of the samples in specialized laboratories. In the last decade, several biosensors (devices based on biological parts that sense a signal and transform it into a quantifiable response) for easy in situ detection of contaminants have been developed (Tecon and van der Meer 2008; Segura et al. 2017). However, the toxicity of the contaminant toward the biosensor (if whole cell biosensors are used) and the possibility of synergistic effects with other compounds in the environment can alter the results making biosensors, in general, not as accurate as chemical methods (Diplock et al. 2010). Nevertheless, robust biosensors with high specificity for oil-derived compounds are available (Kohlmeier et al. 2008; Sevilla et al. 2015; Hernández-Sánchez et al. 2016). When chemical methods are used, the total amount of the contaminant is detected, whereas the biosensors detect the bioavailable fraction; this is important as the bioavailable fraction determines the ecotoxicity of the contaminant (Tecon and van der Meer 2008).
4 Improving Rhizoremediation: Research Needs
One important problem for soil bioremediation, whatever technique is used, is the bioavailability of the pollutant. Most organic contaminants are highly hydrophobic compounds that dissolve poorly in water and many can form complexes with soil particles; this lack of bioavailability often lowers removal efficiencies. Soils with a long history of contamination are generally much less responsive to rhizoremediation than freshly spiked soils (Olson et al. 2007). It has been demonstrated that surfactants, in general, can improve the degradation of the contaminants (Ortega-Calvo et al. 2013). However, as they lead to an increase in the bioavailability of the contaminant, the addition of these compounds may produce negative effects on microbial and plant growth during rhizoremediation. Therefore, rhizoremediation techniques have to be carefully designed to improve the bioavailability of the contaminant without affecting growth. Microbial biosurfactants have been widely studied (Marchant and Banat 2012) and commercial biosurfactants are already available; however, their use in rhizoremediation has not been thoroughly investigated (Liduino et al. 2018). The selection of bacteria which are able to produce biosurfactants in the rhizosphere of plants is an interesting alternative for improving the removal efficiency. In this context, Kuiper et al. (2004b) identified bacteria growing in a PAH contaminated area that produce biosurfactants (putisolvins) that facilitate the solubilization of PAHs and hence biodegradation by microorganisms. This property is also of interest because a number of biodegradative microorganisms exhibit positive chemotaxis towards the pollutants. Therefore, the combined action of biosurfactants and chemotaxis can contribute to bacterial proliferation and to microbial spread in polluted soils, in order to clean larger zones. Besides amino acids, sugars, and other small molecular-sized compounds, plant rhizodeposition including mucilage, high molecular compounds, and other substances could improve the bioavailability of contaminants fostering biodegradation by microbial activity. To the best of our knowledge, no studies about the solubilization of hydrophobic contaminants by plant rhizodeposits have been reported.
Advances in knowledge regarding the molecular interactions between plants and microorganisms, in the expression of catabolic genes in the rhizosphere, and in the selection of the best plant-microbe combinations will have to be converted into field strategies that demonstrate the usefulness of this approach. The release of non-native and recombinant microorganisms into the soil is still a controversial subject and more studies to analyze the impact on indigenous microbial communities are needed.
It is important to know the fate of the contaminants during rhizoremediation as the translocation of contaminants to the aerial part of the plant may facilitate their spread through the trophic chain. Adsorption of the contaminants to the root cell wall and/or transport of the toxic chemical into the plant are research topics that have received increasing attention in recent years (Dupuy et al. 2016; Cennerazzo et al. 2017). As the economic cost of bioremediation efforts is one of the main aspects that are being considered by contaminant producing industries and it has been demonstrated that the microbial biodegradation activity in soil favors the optimal growth of the plant (Van Dillewijn et al. 2007; Rodriguez-Conde et al. 2016; Vergani et al. 2017), it will be interesting to study the possibility of receiving direct economic benefits through the cultivation of crops while remediation is taking place. The economics of this new perspective and the safety-related issues regarding the possibility of the introduction of contaminants into the food-chain are areas of research that remain unexplored.
The work by authors was supported by research grants from the Spanish Ministry of Science and Innovation and the Andalusian Regional Government, (Junta de Andalucía). We thank Angela Tate for improving the use of English in the manuscript.
- Böltner D, Godoy P, Muñoz-Rojas J, Duque E, Moreno-Morillas S, Sánchez L, Ramos JL (2008) Rhizoremediation of lindane by root-colonizing Sphingomonas. Microbial Biotech 1:87–93Google Scholar
- Bulgarelli D, Rott M, Schlaeppi K, Ver Loren van Themaat E, Ahmadinejad N, Assenza F, Rauf P, Huettel B, Reinhardt R, Schmelzer E et al (2012) Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 488:91–95. https://doi.org/10.1038/nature11336CrossRefPubMedGoogle Scholar
- Burges A, Alkorta I, Epelde L, Garbisu C (2017) From phytoremediation of soil contaminants to phytomanagement of ecosystem services in metal contaminated sites. Int J Phytoremediation. https://doi.org/10.1080/15226514.2017.1365340
- Burken JG (2004) Uptake and metabolism of organic compounds: green-liver model. In: McCutcheon SD, Schnoor JL (eds) On phytoremediation. Wiley, New York, pp 59–84Google Scholar
- Colleran E (1997) Uses of bacteria in bioremediation. In: Sheehan D (ed) Methods in biotechnology, vol 2: bioremediation protocols. Humana Press Inc., TotowaGoogle Scholar
- Kappell AD, Wei Y, Newton RJ, Van Nostrand JD, Zhou J, McLellan SL, Hristova KR (2014) The polycyclic aromatic hydrocarbon degradation potential of Gulf of Mexico native coastal microbial communities after the Deepwater Horizon oil spill. Front Microbiol 5:205. https://doi.org/10.3389/fmicb.2014.00205CrossRefPubMedPubMedCentralGoogle Scholar
- Kohlmeier S, Mancuso M, Deepthike U, Tecon R, van der Meer JR, Harms H, Wells M (2008) Comparison of naphthalene bioavailability determined by whole-cell biosensing and availability determined by extraction with Tenax. Environ Pollut 156:803–808. https://doi.org/10.1016/j.envpol.2008.06.001CrossRefPubMedGoogle Scholar
- Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GEM, Thomas-Oates JE, Lugtenberg BJJ, Bloember GV (2004b) Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and breakdown existing biofilms. Mol Microbiol 51:97–113CrossRefPubMedGoogle Scholar
- Liu J, Xiang Y, Zhang Z, Ling W, Gao Y (2017) Inoculation of a phenanthrene-degrading endophytic bacterium reduces the phenanthrene level and alters the bacterial community structure in wheat. Appl Microbiol Biotechnol 101:5199–5212. https://doi.org/10.1007/s00253-017-8247-zCrossRefPubMedGoogle Scholar
- Ortega-Calvo JJ, Tejeda-Agredano MC, Jimenez-Sanchez C, Congiu E, Sungthong R, Niqui-Arroyo JL, Cantos M (2013) Is it possible to increase bioavailability but not environmental risk of PAHs in bioremediation? J Hazard Mater 261:733–745. https://doi.org/10.1016/j.jhazmat.2013.03.042CrossRefPubMedGoogle Scholar
- Segura A, Hernández-Sánchez V, Marqués S, Molina L (2017) Insights in the regulation of the degradation of PAHs in Novosphingobium sp. HR1a and utilization of this regulatory system as a tool for the detection of PAHs. Sci Total Environ 590–591:381–393. https://doi.org/10.1016/j.scitotenv.2017.02.180CrossRefPubMedGoogle Scholar
- Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangronsveld J, van der Lelie D (2009) Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microbiol 75:748–757. https://doi.org/10.1128/AEM.02239-08CrossRefPubMedGoogle Scholar
- Uhlik O, Wald J, Strejcek M, Musilova L, Ridl J, Hroudova M, Vlcek C, Cardenas E, Mackova M, Macek T (2012) Identification of bacteria utilizing biphenyl, benzoate, and naphthalene in long-term contaminated soil. PLoS One 7(7):e40653. https://doi.org/10.1371/journal.pone.0040653CrossRefPubMedPubMedCentralGoogle Scholar
- Vergani L, Mapelli F, Zanardini E, Terzaghi E, Di Guardo A, Morosini C, Raspa G, Borin S (2017) Phyto-rhizoremediation of polychlorinated biphenyl contaminated soils: an outlook on plant-microbe beneficial interactions. Sci Total Environ 575:1395–1406. https://doi.org/10.1016/j.scitotenv.2016.09.218CrossRefPubMedGoogle Scholar