12 Role of microbes and plants in remediation – Rhizoremediation

Dr. Yogalakshmi K N

Rhizoremediation Objectives:

To understand the concept of rhizoremediation
To identify suitable plant and microorganism for rhizoremediation
To differentiate the merits and demerits of rhizoremediation
To study about the factors that can improve rhizoremediation

1.Introduction

Natural and anthropogenic activities have led to contamination of natural resources such as soil, air and water. Continuous discharge of pollutants has led to its accumulation in different spheres of the environment. The pollutants can be of different types: organic pollutants, inorganic pollutants and heavy metals. The organic pollutants are released from anthropogenic activities unlike inorganic pollutants and heavy metals that are released from both the sources. The significance of these pollutants was realized subsequent to the environmental and health issues generated by them. Henceforth, it is essential to remove the pollutants from the environment. Soil being an important component of the ecosystem serves as a sink of all pollutants. The clean-up of soil is a challenging job and can be achieved through remediation. Physical and chemical remediation is not feasible as they are mostly ex-situ technologies. However, biological remediation or bioremediation is a good clean up technique as they can be performed onsite or insitu. Bioremediation is a process that uses living organisms, mostly microorganisms and plants, to degrade and reduce or detoxify waste products and pollutants. The bioremediation technologies can be sub categorized into bioremediation, phytoremediation and rhizoremediation according to the biological agents used. Bioremediation is the remediation technology which uses microorganisms such as bacteria, fungi and actinomycetes for degradation or removal of the pollutant from the contaminated soils. Phytoremediation utilizes green plants for removal of the pollutants. Whereas, rhizoremediation is the microbe-plant assisted remediation approach to clean the contaminated soil. Rhizoremediation is gaining more attention since last decade. The selection of bioremediation or phytoremediation for cleanup of a contaminated site may depend upon prevailing conditions that support the application of microbes, plants, and/or both. Without the microbial contribution, phytoremediation alone may not be a viable technology for many hydrophobic organic pollutants (Chaudhry et al., 2005).

2.Rhizoremediation

Rhizoremediation or rhizosphere bioremediation is the biological remediation approach where microorganisms degrade or remove the soil pollutants in the rhizosphere by utilizing the amazing metabolic capabilities of rhizospheric microorganisms and plant roots. The term consists of both stimulation and rhizodegradation thus, describing the the plant and microbes beneficial interaction. The association between plants and bacteria is an interesting area of research which got special attention in the last decade. The term “Rhizosphere” was first coined by Hiltner in 1904. He described it as the zone of soil adjacent to the plant root system. The rhizosphere provides an active microenvironment, where microorganisms with plant roots, form exclusive communities that have significant potential for detoxification or degradation of hazardous substances.

Rhizoremediation technology involves the following steps: 1) Identification and selection of the microorganism, 2) selection of the polluted site, 3) enrichment of the selected microorganism in the concerned pollutant, 4) inoculation of the enriched microorganism to the plant and root colonization, and finally 5) microbe-plant host interaction that can be used to clean that contaminated site. Kupier et al. (2002) described rhizoremediation as a successful technique for PAH contaminated soil and is depicted in Figure 1. Briefly, bacteria were isolated from the roots of grasses grown in PAH (polycyclic aromatic hydrocarbon) contaminated soil. Bacteria were isolated and enriched in naphthalene medium and selected for colonization with the roots of the plant selected.

3.Selection of plant and microorganisms for rhizoremediation

Selection of the micro-organism and plant is an important and crucial step for deciding the success of rhizoremediation. From the studies, it is suggested that grasses and legume plant species are most suitable for rhizoremediation as these plants posses highly branched root system and can harbour a large population of micro-organisms. Microorganism selection too plays a significant role in rhizoremediation. Rhizosphere microorganisms are considered as most suitable microorganisms for rhizoremediation because these microbes posses root growth stimulating or inhibiting properties. The ideal plant or microorganism should possess the following characteristics.

Plant selected for rhizoremediation should possess the following characters:

Fast growth and high biomass: The plant should grow will even under pollutant stress conditions. The pollutant should not hinder the growth of the plant.
Widespread highly branched root system: The plant should have a profuse, well branched and branched root system. This will facilitate the habitation of more rhizosphere microorganisms.
Ability to accumulate metals preferably in the aboveground parts.
Tolerance to metal concentration accumulated: The plant should be resistant to the pollutant and withstand the varying pollutant concentration.
Easy to harvest
Non consumable by humans and animals: Non edible plants are generally preferred for rhizoremediation to avoid health hazards to living beings. This will also avoid the entry of pollutant into the food chain.

Microorganism selected for rhizoremediation should possess the following traits:

The selected microorganism must have capability to establish interacting relationship with the plant selected for rhizoremediation
Microbes must be able to proliferate in the root system
Catabolic pathways must be operative
Microorganism must possess plant growth promoting factors

Further success of rhizoremediation also depends on other factors such as establishment, primary and secondary metabolism, ecological interaction with other organisms and survival.

4.Rhizosphere and associated microorganisms

Hiltner described the rhizosphere ‘as the area around a plant root that is inhabited by a unique population of microorganisms influenced by the chemicals released from plant roots’. Rhizosphere consists of three zones classified based on their relative proximity and their influence on the root. The cortex and endodermis consists of the endorhizosphere. It is place where microbes and cations occupy the “free space” between cells called the apoplastic space. The rhizoplane is the medial zone directly adjacent to the root including the root epidermis and mucilage. The outermost zone is the ectorhizosphere which extends from the rhizoplane out into the bulk soil. The size and shape of rhizosphere is not definable. It is highly complex and consists of a variety of microorganisms. The chemical, biological and physical properties of the rhizosphere change both radially and longitudinally along the root.

The Rhizosphere is inhabited by a diverse range of microorganisms and is zone of high metabolic activity. In addition to the microorganisms, an array of compounds such as enzymes, sugars, amino acids, organic acids, vitamins, and esters collectively called the root exudates are also present in the rhizosphere. Other secretions from the micro-organisms are also present in the rhizosphere. It is confirmed from the studies that rhizosphere soil contains large number of microbial population as compared to the bulk soil. Rhizosphere microbial population type and size can be determined by various factors such as soil type, plant species, age of the plant, root exudates, type and concentration of the pollutant. The rhizosphere biota includes the free living root associated bacteria, symbiotic bacteria and mycorhizal fungi that colonize the root environment. The Rhizobiota use these root secretions as carbon and energy source and promote host plant growth and protect the plant from pollutants and pathogens (Toussaint et al., 2012).

4.1 Bacteria

Rhizobacteria with plant growth promoting, soil contaminant degradation potential and defence properties are characterized as plant growth promoting rhizobacteria (PGPR). PGPR can be symbiotic (i.e.) present intracellular between the root cortex cells (iPGPR) or free-living existing extracellular (ePGPR) in the rhizosphere (Khan, 2005). Symbiotic bacteria invade plant cells (e.g.) root nodule bacteria such as Bradyrhizobium, Mesorhizobium and Rhizobium while free living rhizobacteria remains outside of plant cell (e.g.) Agrobacterium, Arthrobacter, Azotobacter, Micrococcous, Pseudomonas, Bacillus and Serratia (Gray and Smith, 2005). Many rhizobacteria do exhibit unusual traits to live under stress conditions such as drought, salinity and heavy metal pollution (Wani and Khan, 2010). In recent years such rhizobacteria growing under stressful environment are more explored in rhizoremediation.

Rhizosphere is mainly dominated by gram-negative bacteria, Pseudomonas and several studies have confirmed bacterial heavy metal resistance, isolated from diverse habitats. So worldwide rigorous research is going on to explore rhizobacteria with novel traits such as metal detoxifying potentials, pesticide degradation, salinity tolerance, pathogens and insects control besides other plant growth promoting traits such as phytohormone, siderophore production, 1aminocyclopropane- 1-carboxylate, hydrogen cyanate (HCN), nitrogenase activity and phosphate solubilisation ability etc.

4.2 Fungi

Along with bacteria, rhizosphere is also inhibited by fungi that posses good potential for rhizoremediation. Rhizosphere is mainly dominated by fungal genera namely Aspergillus, Fusarium, Penicillium and Verticillium. The zoospore forming fungi such as Aphanomyces, Pythium and Phytophthora are chemotactically attracted towards roots by the chemical compounds secreted by the roots. Likewise, several fungi namely Gibberella and Fujikurio produe phytohormones, that stimulate the plant growth.

4.3 Actinomycetes, protozoa, algae

In the rhizosphere actinomycetes, protozoa and algae have not been studied for rhizoremediation potential. It is generally understood that the actinomycetes are less stimulated in the rhizosphere than bacteria. However, when antagonistic actinomycetes increase in number they suppress bacteria. Generally, actinomycetes increase in number when antibacterial agents are sprayed on the crop. Among the actinomycete, the phosphate solubilising namely Nocardia, Streptomyces have a dominant role to play. Due to large bacterial community, an increase in the number or activity of protozoa is expected in the rhizosphere. Flagellates and amoebae dominant the region unlike ciliates which are rare in the region.

5.Mechanism of rhizoremediation

There are three separate and interacting components of the rhizosphere:

Rhizosphere (soil): the zone of soil influenced by roots through the release of substrates that affect microbial activity.
Rhizoplane: the root surface, including the strongly adhering soil particles.
Root tissue: that some endophytic microorganisms (endophytes) are able to colonize.

The varying physical, chemical, and biological properties of the rhizosphere soil, compared with those of the surrounding soil, are responsible for changes in microbial diversity, number and metabolic activities of microorganisms in the rhizosphere microenvironment, the phenomenon called the rhizosphere effect. Microbial populations in rhizosphere soil affect the heavy metals mobility and availability to the plant, and degradation of other hydrophobic organic pollutants by various mechanisms such as release of chelating agents, acidification, phosphate solubilization, and redox changes (Smith and Read, 1997).

6.Improving rhizoremediation efficiency

The biodegradative capabilities of the microorganisms and expression of the requisite microbial genes in the rhizosphere are very much important for the rhizoremediation. However, some other aspects can also improve the efficiency of the process. Such aspects are described below.

6.1 Selection of the best plant-microbe combination

As mentioned in the previous section, the composition of root exudate in the rhizosphere changes with the different plant developmental stage and plant species. The variations in exudation can evidently impose different effects on the rhizospheric microbial community. Similarly, the root exudates composition in the rhizosphere also favours the proliferation of microbes that degrade them efficiently. For example flavonoids are secreted by plants as a defence mechanism against pathogens and plants with higher amount of flavonoids can be efficiently colonized by tolerant microbial population.

The environmental stress in the rhizosphere also varied the quality and quantity of the root exudates. Nutrient and water stress increase the root hair density likewise, phosphorus deficiency causes an increase in exudation secretion due to reduction in integrity of the membrane. These stresses resulted in increase in rhizobacteria population (Chaudhry al. et, 2005). However, higher concentration of the contaminant can also affect the root growth and hence decrease or diminish the biodegradation of the contaminant. Therefore, use of bacteria with plant growth promoting factors (PGPF) can overcome this problem. In recent times rhizobacteria with plant growth promoting factors attracted much attention in rhizoremediation.

Similarly, root exudation quality and quantity determine the fate of microbial population in the rhizosphere. Rhizospheric microbial population also helps in promoting or inhibiting the plant root growth by making the rhizosphere environment clean through degradation of the contaminant.

6.2 Endophytic bacteria

Rhizobacteria are the most commonly used microbes in the rhizoremediation. However, in some recent studies, application of endophytic bacteria in rhizoremediation proved to be more efficient and advantageous due to following characters possessed by the endophytic bacteria:

1) Endophytic bacteria inhibit the interior tissues of the plant and get protection from the direct contact with the contaminant present in outside environment.

2) Causing no negative effect on the host plant due to non-competitive nature for nutrients.

3) It is easy to isolate an endophytic bacterium from the host plant, enrich in the requisite contaminant and re-inoculate the bacteria to the same plant for bioremediation.

Therefore, application of endophytic bacteria in bioremediation has been studied in last years (Doty et al., 2008). Endophytic bacteria are inoculated by various methods including seed inoculation, pruned-root dip, soil drench and foliar spray. The choice of endophytic bacteria inoculation method depends on the specific type of plant-endophyte combination to be used in rhizoremediation. Endophytic bacteria from family Pseudomonaceae, Burkholderiaceae and Enterobacteriaceae are commonly used in rhizoremediation. The endophytic bacteria are not yet studied completely and are at infant stage. Some reports are available on degradation capability of endophytic bacteria on hydrocarbons, herbicides and explosives. Some studies also reported high tolerance of endophytic bacteria to heavy metals, BTEX (benzene, toluene, ethyl‐benzene and xylenes), TCE or PAHs.

6.3 Seed colonization

The efficiency of rhizoremediation can be improved by using the appropriate microbial inoculation method. One of the least expensive technique of microbial inoculation is to cover the seeds with the appropriate microorganism. For this, the microorganism used should posses the character of adherence to the seed (Colleran, 1997). The microbial cell adhesion to the seed has been studied by using viable cell counts and through reporter genes namely, gfp or lux genes. Microscopic techniques and Scanning electron microscopy (SEM) has been used to track the microbial adherence to seeds. The mechanism of attachment of microbes especially bacteria to the biotic surfaces such as seeds and roots is common.

6.4 Production of biosurfactants

Rhizoremediation efficiency can be increased by increasing the bioavailability of the contaminant. Most of the organic pollutants are hydrophobic and are not able to dissolve in water. These organic pollutants form insoluble complexes with the soil particles and are biologically unavailable to the bioremediating organism. Microorganisms use different approaches to promote the bioavailability of hydrophobic pollutants. These approaches include secretion of extracellular polymeric substances, biosurfactant production and biofilm formation on the pollutant surface. Biosurfactants are amphiphilic in nature and form spherical or lamellar micelles when the surfactant concentration exceeds a critical micelle concentration that is specific for each compound. Hydrophobic contaminants become solubilized in the hydrophobic cores of the micelles, which increases the transfer of the compounds from a solid to water phase where it becomes more accessible to bacteria. One important group of bacterial biosurfactants are the glycolipids of which rhamnolipids are the major representative. It has been shown that rhamnolipids are able to enhance the biodegradation rate of contaminants. Kuiper and colleagues (2004) isolated a Pseudomonas putida strain from plant roots at a site polluted with PAHs that produce two lipopeptide biosurfactants. These lipopeptides (named putisolvins) increased the formation of emulsions with toluene. Searching for rhizobacteria that promote the bioavailability of contaminants is therefore of great interest in the context of bioremediation. This property is also of interest because a number of biodegradative microbes exhibit positive chemotaxis towards the pollutants (Parales and Haddock, 2004). Therefore, the combined action of the biosurfactant and chemotaxis may contribute to bacterial proliferation and microbial spread in polluted soils, leading to the clearing of more ample zones.

6.4 Production of biosurfactants

6.5 Engineering bacteria for rhizoremediation

Application of genetically engineered microorganisms in increasing bioremediation efficiency has become a common approach in recent times. Rhizoremediating microorganisms can be genetically modified by introduction of catabolic genes, construction of hybrid pathways, promoter modification for increased expression of genes of interest and construction of recombinant strains. Recombinant strains are the strains that posses two or more trait in combination such as degradation of the contaminant, production of biosurfactant, good root colonization capability and plant growth promoting factors etc.

However, still the release of recombinant microorganisms in the field is restricted in many countries and these legal limitations, together with some well sustained scientific concerns, may limit the development of this field

7.1 Merits and demerits of rhizoremediation

Like any other remediation technologies rhizoremediation also possess some merits and demerits.

7.1 Merits of rhizoremediation

Rhizoremediation possess the following advantages over other biological remediation technologies and conventional physical and chemical remediation technologies.

It is an in-situ technique
It is more efficient than other biological remediation technologies
It requires less expenditure and energy
It is natural and eco-friendly in nature
Rhizoremediation takes advantage of plant roots natural symbiosis with mycorrhiza and root associated natural microbial flora for the enhanced degradation of pollutants in the rhizosphere.
It is easy to execute in field conditions Easy to monitor
Further the application of genetically modified organisms in bioremediation can increase the efficiency of rhizoremediation

7.2 Demerits of rhizoremediation

It is a slow process and requires days to months for bioremediation of any pollutant For in-situ rhizoremediation soil must be of high permeability
It does not clean the site completely, some concentration of the pollutant remains even after the process
Still the mechanism of interaction between plant and microbe is not fully explored

Application of rhizoremediation

It is useful for degradation of organic pollutants
It can help the accumulation of heavy metals in plants
It is useful for remediation of contaminated soils on large scale

Summary

At the end of this module we have studied about:

Definition and concept of rhizoremediation
Mechanism of pollutant removal by rhizoremediation
Merits and demerits of rhizotoremediation
Strategies used to improve the efficiency of rhizoremediation

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References

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https://www.nature.com/scitable/knowledge/library/the-rhizosphere-roots-soil-and-67500617
http://plantsinaction.science.uq.edu.au/content/421-rhizosphere