METHODS FOR BIOREMEDIATION OF METAL CONTAMINATED SOILS
Physiochemical analysis of soil is a crucial step while designing a bioremediation protocol for any site. The efficiency of phytoremediation and microbial remediation depends on the physical and chemical state of native soil. Various parameters are evaluated to check the quality of the soil. Physical indicators include water holding capacity, density, and texture. The chemical analysis involves pH, conductivity, C, S, P levels, and biological properties include microbial biomass, including mainly nitrogen and carbon (Gil-Sotres et al., 2005; Nanda and Abraham, 2013). The enzymatic activities are also analyzed to check the status of nutrient cycling, and it includes an assay of mease, phosphatase, and p-galactosidase. The gr owth of plants and the activity of microbes are highly influenced by the pH of the soil. The presence of metal ions is also affected by the local conditions of the spoil. With the decrease in pH, solubility of metal ions was found to increase (Sanders and Adams, 1987).
Phytoremediation is the use of plants for the removal of pollutants from contaminated sites. Worldwide research has been carried out in this area involving a large number of plant species. Among all the plant species, plants belonging to the categoiy of hyperaccumulators showed promising results. Hyperaccumulators are those species that have the ability to accumulate certain chemicals, heavy metals at very high concentrations without themselves being harmed. No impact of such elevated level of heavy metal on the growth of these plants. The hyperaccumulators can be distinguished from nonhyperaccumulators type based on mainly three properties, i.e., increased uptake of heavy metals, rapid translocation from root to shoot (Rasico and Navari-Izzo, 2011) and more detoxification rate of heavy metals via leaves of such plants. The plants belonging to the Brassicaceae family have been the most widely exploited species used for their phytoremediation potential for the removal of heavy metals from different sites (Sims, 2006; Perfumo et al., 2007; Ahmad, 2017). Species belonging to Brassica, Arabidopsis, Bommuellera, Alyssum have been reported to be successful hyperaccumulators (Ahmad, 2017). In some plants, metal sequestrations have found to be carried out by the presence of specific phytochelators (Cobbett and Goldsbrough, 2002). Metallothioneins are the other classes of molecules responsible for the successful removal of metal ions from the contaminated sites. Organic fertilizers were considered as a better source of nutrients for plants due to their nontoxic behavior. There were several points that make them popular as compared to chemical fertilizers. But recent studies have reported their role in augmenting the natural microflora of the soil (Masto et al., 2006). The carefiil selection of biofertilizer would further help in increasing the efficiency of bioremediation. Juwarkar et al. (2008) reported the use of Azotobacter resulted in an increased rate of removal of heavy metal from the soil as compared to the initial studies where plants were used alone for the removal of contaminants.
Plants employed various mechanisms for the removal and immobilization of heavy metals in the soil. Phytotransfomiation and phytodegradation have been successfully reported by many plants in which certain enzymes carried out the conversion or breakdown of potential pollutant into nonreactive form (McCutcheon and Schnoor, 2003). Rliizofilteration is another approach where contaminants are absorbed or precipitated or absorbed by the action of root exudates and enzymes on the surface of roots (McCutcheon and Schnoor, 2003).
The increased solubility resulted in increased bioavailability and hence, enhanced uptake by the plants and microbes. The results of the enzymatic analysis can be used as indicators of not only the biogeochemical cycling of nutrients but also the index of microbial activity. The enzymes present in soil are mainly contributed by the microbial population (Ladd, 1978), followed by plant and animal origin (Tabatabai, 1994). The concentration and solubility of heavy metals affects the enzymatic activity of various microbes by influencing the availability of substrates, feedback inhibition, and chemical inhibition. On the other hand, some metals act as cofactors and in turn, enhanced the activity of certain enzymes (Nanda and Abraham, 2013). Hence the addition of biofertilizers can be used to increase the bioremediation potential of both the microbes and plants. The sludge from various sources like dairy sludge, effluents having high organic matter content, and metal content can act as a potential source of organic nitrogen, phosphorus, and carbon and also act as a source of microbial stock (Juvarkar et al., 2008).
Phytoextraction is another technique based on the use of plants for the removal of trace elements from the soil. It is based on translocation of elements into the harvestable part of the plant followed by post-harvest treatment of the recovered biomass by employing methods like thermal treatment and composting for reducing the biomass volume and recovery of trace elements from the selected plant (Vangronsveld et ah, 2009). But the main disadvantage of the above method is the longer time period required for the growth of plant and bioavailability of the element for translocation to the harvestable part of the plant. Vangronsveld et ah (2009) reported the use of phytoextraction potential of hyperaccumulators like Thlaspi sp. and Alyssum sp. for the removal of trace elements. In a study earned out by Kunito et ah (2001) in copper contaminated sites in rhizospheric and non rhizospheric area of Phragmites, significant difference in the bacterial communities was observed. Rhizospheric bacteria were observed to produce certain exopolymers that bind to trace elements and thus facilitates their translocation into plants. Copper resistant Bacillus sp. dominated the rhizosphere region of Phragmites, while the non-rhizospheric region was dominated by Methylobacterium sp. (Kunito et ah, 1997). In another study earned out by Mengoni et ah (2004) using a cultivation-independent approach, Proteobacteria dominated the rhizospheric area of A. bertolonii, while alpha and gamma proteobacteria dominated the leaf community of the plant. The culture-dependent approach reported different communities of bacteria related to genera Bacillus, Staphylococcus, Microbacterium in A. bertolonii (Barzanti et ah, 2007). Thus both culture-dependent and independent studies should be studied together to have a better understanding of the bacterial communities associated with these types of plants. This will further help in designing protocols for bioremediation.
It is a method being employed to reduce the bioavailability of heavy metals in the soil by erosion. It involves the use of plants to trap and hold heavy metals and not allowing their movement through the soil, thus reducing their cover zone and also preventing their leaching into groundwater (Salt et al., 1995; Chubuike and Obiora et al., 2014). It is an alternative method to phytoextraction (McGrath and Zhao, 2003). It has been observed that in phytostabilization, plants convert more toxic form of heavy metals to a less toxic form and immobilize them using their roots and thus preventing their movement in the soil and contaminating it further (Wu et al., 2010; Ali et al., 2013). Continuous studies are being performed to tiy and test new plant species which could prove to be a better phytostabilizer plant. One such exploration is being done with Vossia cuspidate and macrophtye. Galal et al. (2017) used this plant to against common heavy metals including chromium, copper, lead, and aluminum. They found accumulation of these metals in their root and shoot system and thus preventing their leaching into soil and thus prove to be a good phytostabilizer. This method can be used in combination with microorganisms which improves their efficiency. For example, Ahsan et al. (2017) make use of a combination of plant and bacteria for remediation of soil contaminated with lead and uranium. In this approach, they used different bacterial endophytes, including Enterobacter sp. HU38, Microbacterium arborescens HU33 and Pantoea stewartii ASI11 in combination with Leptochloa fusca. Results showed an enhance growth of plant in contaminated soil due to bacterial endophytes. Not only bacterial endophytes help in growth enhancement but also increased the metal uptake by plants and thus stabilizing the soil. Phytostabilization as a method for remediation of the heavy metal contaminated environment has proven its efficiency, but as it only limits the movement of heavy metals in the environment and does not remove, it can be a problem.
It is a type of method involving plants that can uptake heavy metals from the contaminated soil, converting them into volatile form and then releasing them into the atmosphere (He et al., 2015). This method has been mostly employed for the treatment of mercury-contaminated environment in which using specific plant species, mercuric ion is converted to elemental mercury (US Environment Protection Agency, 2000). Making the method more efficient genetic engineering has been used to develop transgenic plant species to treat contaminated soil. These are Arabidopsis thaliana, Nicotiana tabacum (Rugh et al., 1998; Meagher et al., 1999; Chubuike and Obiora, 2014). The problem with this method is that the metal contaminant is only converted from one form to another and is not completely removed from the environment; ultimately, the metal remains in the environment, which can be get resuspended into soil by rainfall. Table 9.1 summarizes the different plants being used to bioremediate heavy metal contamination in soil.
TABLE 9.1 Different Plant Species Being Used to Treat Heavy Metal Contamination in Soil
Microorganisms can be used as an effective agent for cleaning up the contaminated sites. A wide variety of microorganisms can be isolated from extreme regions of the environment. As they are continuously exposed to these extreme conditions, they developed certain unique features which can be exploited for various purposes. In the same way, microorganisms residing in polluting sites have more potential to degrade the novel xenobiotic compounds, which are otherwise difficult to degrade by other means. The novel microorganisms can be developed in laboratory with the use of genetic engineering as per the requirement of the particular site such microorganisms, which are popularly known as GEM (genetically engineered microbes) have more potential and better survival and success rate as compare to naturally isolated microbes. One such successful use of superbug Pseudomonas putida engineered by Chakrobarty showed a high success rate in removal of oil spills. Various techniques are available for introducing microbes to the rare of contamination. The best approach to add consortia rather than single microbe as the former had a better survival rate due to efficient competition they could give to native microflora without themselves getting inhibited due to limitation of nutrients and other environmental factors. As already discussed, bioaugmentation, i.e., addition of microbes from outside into the site, biostimulation which is the addition of nutrients to stimulate the activity of already occurring microbes and some of the famous approaches being used for in situ bioremediation. Biosorption is another successful technique employed in bioremediation where metal species are adsorbed or absorbed by the microbial cells. Depending on the type of microorganism and metal species, metal may be absorbed or precipitated by the action of membrane transport pumps, enzymatic actions, or they may be translocated to specific sites in that organism (Gadd, 1996). Li et al. (2013) reported the successful use of microbes in sequestration of heavy metals from highly stable states. The microorganisms along with plants have more potential for remediation as compared to that case when both approaches are being used alone. The rhizosphere is the region where majority of the microbes interact with the plants. Most of the nutrients and minerals are taken by the plant from the rhizosphere area. Hence the region has the high potential to be explored for the bioremediation. The roots of the plants secrete variety of exudates ranging from organic acid, sugars, vitamins, enzymes to siderophores. The rhizospheric microbes have synergistic associations to increase the bioavailability of certain nutrients by the microbial action which in turn get the nutrients from the plants (Barea et al., 2002; Yang et al., 2009). Varieties of metal chelators are secreted by the roots of higher plants. Such chelators facilities the release of metal ions bounded to the soil particles by forming complexes with them and hence increased their bioavailability. Dakora and Philip (2002) reported the use of phytochelators for removal of heavy metals from the soil. The plant growth-promoting bacteria (PGPB) popularly known as PGPR have been widely studied and known to promote gr owth of the associated plants. Several mechanisms are responsible for the action including competition for binding site in the root region, production of cell wall lysing enzymes and antibiotics (Glick et al., 1995; Glick et al., 2007).
Apart from promoting the growth of plants by the above mechanisms, they are also reported to be effective biocontrol agents. The attention is now diverted for exploring the application of these rhizobacteria in removal of contaminants from the soil. The degradation pathway of polychlorinated biphenyls and the genes responsible for them were well studied in bacteria isolated from the rhizophoric region of the plants. The only lacunae observed in this case were loss of activity of these bacteria under nutrient-deficient conditions (Brazil et al., 1995; Nonnander et al., 1999). Endophytic bacteria are other bacteria with a great potential to facilitate the removal of heavy metals by increasing the rate of phytoremediation in host plants. In a study earned out by Idris et al. (2004) in Thlaspi goesingense, a plant known for its nickel hyperaccumulation, endophytes showed higher resistance to elevated levels of heavy metals as compared to rhizobacteria. The major problem associated with the use of endophytes in bioremediation programme is that most of they are non-culturable. The more studies are required in this area to explore the actual mechanism, involved in the role of endophytes in promoting accumulation of metals in plants at such a high concentration (Weyen et al., 2009). Besides using bacteria for remediation studies, mychorrhizae was also reported for then potential to be used for removal of pollutants from the soil. They were known to provide resistance to the host plant from the toxic effect of pollutants by forming protective sheath around the plant roots and also affect the transport of pollutant by modifying the soil moisture content (Mehrag and Caimey,
Due to the limitations of methods like phytostabilization and phytovolatilization, new methods are being taken into consideration. One such method is the use of biochar. It is a carbon-rich organic compound produced by pyrolysis of biological material such as wood, manure, and feedstock. Biochar increases the soil pH due to which the mobility and bioavailability of heavy metals in the soil decreases, and also their uptake by the plants also decreases (Ferreiro et al., 2013; Ahmad et al., 2014). In addition to this biochar uses several other mechanisms to decrease mobility or prevent the uptake of heavy metals by plants including complex formation of heavy metals and different chemical groups present in biochar, exchange of heavy metals with the cations present in biochar, electrostatic attraction between positively charged heavy metals and negatively charged biochar components or vice versa (Uchimiya et al., 2011).
TABLE 9.2 Common Contaminants in Soil and Microorganisms Being Used to Treat Them
It has been well reported that Biochar decreases the bioavailability for plant uptake of certain heavy metals like cadmium, arsenic, and lead in soil (Namgay et al., 2010). Lu et al. (2017) conducted a research to study the effect of biochars produced from bamboo and rice straw on heavy metals like zinc, cadmium, copper, and lead. Biochars from both bamboo and rice straw were applied at different concentrations on sandy loam soil contaminated with above-mentioned heavy metals. It was found that there was a decrease in the mobility of the heavy metals with both the types of biochars. The decrease can be linked to the increase in soil pH by application of biochar. However, the effect of biochar on metal mobility varies with the type of biochar applied. Biochar applied for removal of metal of one type may not be that much efficient in removing the other type of metal. In a study, two types of biochars were used, one chicken manure and other from green waste. It was found that the former was effective in reducing cadmium and lead but no significant effect was observed in case of copper whereas the latter significantly reduced the concentration of all the heavy metals, i.e., cadmium, lead, and copper (Park et al., 2011; Ferreiro et al., 2013). Table 9.3 summarizes the different types of biochars used against different heavy metals.
TABLE 9.3 Types of Biochar Being Used for Remediation of Heavy Metal Contaminated Soils