Endophytic Phytoaugmentation: Treating Wastewater and Runoff Through Augmented Phytoremediation

Endophytic Biology

Plants are colonized by a range of microflora such as bacteria, fungi, yeasts, viruses, and protists, as well as epiphytes including algae and nematodes. Plant-microbe populations are dynamic, and variations in microbial communities that are influenced by the large fluctuations in the physical and nutritional conditions, as well as other biotic and abiotic influences, have been observed. ^{2,27 –32} Some microorganisms, predominantly bacteria and fungi, are recruited to enter the plant locally and systemically as endophytes, establishing asymptomatic or mutualistic relationships. ^2,20,33,34 Endophytes are found systemically in roots, stems, leaves, seeds, fruits, tubers, ovules, and some nodules. ^2,35,36 Endophytes may be recruited to their host through chemotaxis, electrotaxis, or simply accidental encounter and are most commonly recruited from the roots. ^31,37,38 Roots have been shown to have the highest localized concentration of endophytes, and endophytic densities tend to decrease from stem to leaf. ^39,40 The most commonly reported endophytic locations are the intercellular spaces and xylem vessels. ⁴¹ Endophytic communities depend on the taxa within a given community, host genotype and corresponding host developmental stage, inoculum density, temporal and seasonal conditions, plant location, and environmental conditions. ^14,17,42,43 Though dynamic, many endophytic communities have been shown to contain common soil taxa such as Pseudomonas, Burkholderia, Bacillus, and Azospirillua. ^37,44

Diversity and Function of Endophytes

Endophytes have been isolated from a diversity of plants, yet their exact function and associations remain unclear. ³⁸ For instance, it is unclear how endophytes interact with each other in the plant, and little is known about the complex endophyte-host molecular interactions or their gene regulation and expression. ^28,41 Endophytes seem to form diverse and complex associations with their hosts, including mutualistic and symbiotic relationships. ^{5,45 –48} In many cases, endophytes are believed to be beneficial to their plant hosts through nitrogen fixation, accelerated seedling emergence, protection from environmental stressors, enhanced nutrient availability and vitamin supply, and contaminant protection and removal. ^9,28,31,37 Endophytes are capable of producing bioactive compounds associated with increased plant growth and health, and offer protection from abiotic and biotic stresses. ^{7,8,37,38,49 –55} Endophytes may also offer their plant host protection and defense against microbial diseases, insects, and nematodes. ^{19,25,33,38,56} For example, endophytic actinobacteria offer defense against the pathogenic fungus Gaeumannomyces graminis in wheat and potatoes, and the endophyte Curtobacterium flaccumfaciens protects citrus plants from the pathogen Xylella fastidiosa. ³⁴ Interestingly, even some mycorrhizal fungi themselves have endosymbiotic bacteria for protection. ^{52,57 –60} More in-depth endophyte reviews are provided by Newman and Reynolds, Sturz et al., Strobel and Daisy, Mercado-Blanco, Tadych and White, and Lodewyckx et al. ^{32,44,61 –64}

Endophytic Augmentation

Many endophytes have shown a natural capacity for xenobiotic degradation. ⁹ Further, plant-associated microorganisms capable of direct degradation are more abundant among endophytes than in the rhizosphere at contaminated sites. ⁶⁵ This may be because the plants themselves selectively enrich degraders inside and around the phytoremediating host plants. Siciliano et al. found that plants grown in soil contaminated with xenobiotics naturally recruit endophytes with contaminant-degrading genes. ³⁰ This selective enrichment suggests a potential against a wide-range of contaminants and sites. ^{26,30,31,65 –67} The natural ability of some endophytes to degrade xenobiotics has been investigated with regard to improving phytoremediation efficiency (Table 1). ^{7 –9,18,20,27,38,51 –53,65,67 –79}

An advantage of using endophytic degraders in remediation is toxic xenobiotics may be degraded in planta, reducing phytotoxic effects and eliminating any toxic effects on herbivorous fauna residing on or near contaminated sites. ^4,9,15 More specifically, some highly water-soluble and volatile organic xenobiotic compounds are quickly absorbed and may remain in the xylem for up to two days, allowing endophytic detoxification. ^6,8,37,63,80 When plant species and environmental conditions are relatively constant, contaminant uptake depends on the lipophilicity, or the log of the octanol-water partition coefficient (log K_ow). ⁷ Relatively hydrophilic compounds, or compounds with a log K_ow between 0.5–3.5, such as benzene and toluene, tend to be absorbed rapidly into the plant. ^{7,45,48,81,82} Once absorbed into the plant, there are several kinetic processes for phytoremediation that may occur, including uptake, transformation, and stabilization via immobilization, which are thoroughly reviewed elsewhere. ^1,5,7,13 When the log K_ow is outside of the uptake range, it may still interact with the plant-microbe system through other methods like adsorption or volatilization. Further, mixed inoculations consisting of more than one endophytic bacterium that inhibit plant growth individually may result in plant-growth promotion, suggesting some important but poorly understood in planta interactions. ³⁸ Consequently, full characterization of the plant-microbe interactions and their effects on pollutant degradation is needed.

Candidates for Endophytic Phytoaugmentation

In general, phytoremediation-based treatment approaches have gained attention for their ecological and economic benefits. Along with site remediation, phytoremediation provides the additional benefits of a high level of public acceptance, pleasing aesthetics, use of naturally occurring plant processes, enhancement of soil and plant health, and improved wildlife habitats. ^1,5,7,53

Several plant species have been tested in phytoaugmentation applications. Poplars (Populus spp.) and willows (Salix spp.) are commonly used because of their rapid growth, deep roots, and large uptake of water. ^83,84 Along with trees, recent reports suggest that hyperacculumators (plants that can accumulate high levels of toxins) and their associated microbes may play a key role in phytoremediation and can support organized endophytic communities. ^{1,5,12,45,64,85} Hyperaccumulators may be used to accumulate heavy metals often found in wastes and at contaminated sites such as As, Cu, Pb, Se, and Zn. ^86,87 This is critical as metals are difficult to remove using traditional techniques and are often only controlled or immobilized. Phytoremediation offers the advantage of metal extraction without disturbing the site. For example, Thlaspi caerulescens (Alpine pennycress) is commonly used to accumulate large amounts of zinc and cadmium, and Thlaspi goesingense is able to accumulate large amounts of nickel. ^{88 –90} The metals may then be reclaimed and reused. ^23,91

Along with hyperaccumulators, some other plants are capable of producing root exudates that enhance contaminant desorption, which may make some compounds more bioavailable for the subsequent degradation by microbial communities. ^39,79 In addition, when augmented with endophytes, some plants have been shown to have improved health and growth, increased drought resistance, reduced transplanting shock, increased resistance to pathogens, and lower mortality. ^{5,8,23,25,40,52,76,79} It has been reported that several endophytic taxa from contaminant-exposed poplar trees have the potential to enhance phytoremediation of volatile organics and herbicides. ^{17,52,76,92,93} A review of phytoremediation plant species and their respective endophytes may be found in Mastretta et al. ³¹

Phytoaugmentation has proven feasible for several endophytic species (Table 1). One key example is the inoculation with a Pseudomonas endophyte capable of degrading the organochlorine herbicide 2,4-dichlorophenoxyacetic (2,4-D) to pea plants, reported by Germaine et al. ⁷⁶ The phytoaugmented plants demonstrated increased removal of 2,4-D and showed no 2,4-D acid accumulation in aerial tissues. ⁷⁶ There is potential to phytoaugment with naturally engineered endophytes, or those who have been genetically altered via natural gene transfer or recombinant DNA technology. ⁸ For example, naturally engineered endophytic Burkholderia cepacia strains with a nickel-resistance operon improved phytoremediation and promoted plant tolerance to toluene. ^51,68 Lodewyckx et al. demonstrated that this same engineered B. cepacia endophytic strain increased the nickel accumulation and tolerance of inoculated plants when added to yellow lupine. ⁷⁴ Transgenic, or genetically engineered, trees may also be designed to increase remediation. ^94,95

Though several studies have reported positive findings such as increased remediation potential or improved plant health with pollutant exposure, there are still research and technology gaps to fill before widespread use can be adopted. Many of the current studies have been conducted at lab scale and with controlled parameters; it is unclear how they may translate to the field or large scale use with dynamic environmental conditions. For instance, it is unknown what metabolites will be produced from pollutant degradation during endophytic phytoaugmentation in the field, nor the ecological impact of long-term endophytic colonization. In addition, though some engineered endophytes are transformed naturally, public education and assurance of safety will need to be demonstrated prior to large-scale use. ³² Poorly understood plant-microbe-pollutant interactions before, during, and after endophytic phytoaugmentation are another obstacle, as are the limited tools and techniques for monitoring these interactions.

Propagation of Beneficial Genes Through Horizontal Gene Transfer

Interest in genetically modifying endophytes is increasing because of their enhanced ability to degrade or resist targeted contaminants. The advantages and obstacles to using bioengineered endophytes have been clearly discussed by Weyens et al. ⁴⁶ Recently, there has been a focus on naturally modified endophytes, through horizontal gene transfer (HGT). Taghavi et al. found the degradative plasmid pTOM-BU61 transferred naturally to a number of different endophytes in planta, resulting in more efficient degradation of toluene in poplar plants. ⁵¹ It has also been reported that HGT in planta is likely to be widespread. ⁹ Further, endophytes have been shown to interact and exchange genes with the rhizopheric and phyllospheric bacterial communities through HGT. ⁷ For instance, genetic transfer to enhance phenol degradation in planta and in the rhizosphere has been demonstrated. ⁹⁶ Similar to genetic bioaugmentation, endophytic phytoaugmentation strategies relying on genetic transfer between the exogenous and indigenous strains have an increased likelihood of success, as they do not require the survival of the donor strains. ^{60,97 –100} Though promising, targeted genetic transfer to increase degradation potential is novel, and little has been done at field scale. From a regulatory standpoint, guidelines for endophytic phytoaugmentation are needed, particularly with respect to engineered endophytes.