34 Bioremediation of Heavy Metals

Dr Kiran Bala

1. Introduction

2. Mechanisms involved in Bioremediation

3. Biological Processes involved in bioremediation 

3.1. Physical-Biological-Chemical Mechanism of Bioremediation 

3.2. Molecular Aspects of Bioremediation Phenomenon

3.3. Hyper accumulators to Heavy Metal Tolerance

4. Heavy Metals Tolerance of Plants through Biochemical Pathways

4.1. Phytochelatins (PC) 

4.2. Metallothioneins

4.3. Biosynthesis and Regulation of FQ Synthetase

4.4. Role of Chelate in Phytoextraction 

4.4.1.  Protective Action of Phytochelatin (PC)

5. Tissue Culture Techniques are Applied in the Process of Bioremediation Concluding Remarks

1.Introduction

Industrialization and urbanisation has led to enormous applications of heavy metals. The processing and disposal of products having heavy metals release these heavy metals and ions into soil and water. This not only degrades the quality of water and soil, but also renders them nutrient deficient and unfit to be used for crop cultivation. Consumption of crops cultivated in heavy metals polluted soil can lead to severe health problems.

Filtration, evaporation, precipitation of chemicals, anion-cation exchange, reverse osmosis, oxidation or reduction, specialized membrane based techniques and treatments by combined electrical and chemical means are some of the major techniques employed to remove such heavy metals. These techniques in many cases stand inefficient at concentrations below 100 mg/L. Alternately, certain techniques based on biological means such as biosorption or bioaccumulation are likely seems effective for elimination of heavy metals. Usage of microorganisms and plants provide a suitable option for remediation of heavy metals since they provide a sustainable means to revive the soil conditions. Bioremediation of heavy metals using microbial communities is governed by various factors like composition of the medium, metal type as well as the microbial species.

Many microorganisms have been known to develop various survival strategies like conversion of toxic heavy metals into less toxic forms (biotransformation), absorption of heavy metals by microbial cells (biosorption), assimilation and accumulation inside microbial cell (bioaccumulation) and biomineralization. Such mechanisms can be experimented for on-site or off-site bioremediation.

Heavy metals can be absorbed by microorganisms through two ways, either active absorption (bioaccumulation) and/or passive absorption (adsorption). The lipids, polysaccharides and proteins present in the microbial cell walls provide carboxylate, hydroxyl, amino and phosphate functional groups that can get attached with ions of heavy metals. If we compare the efficiencies of both biosorption and bioaccumulation processes, we can conclude that for board applications, the biosorption process is more efficient in comparison to the bioaccumulation process, since for active uptake of the heavy metals microbes would require nutrients which in turn would increase the oxygen demand (both biological and chemical oxygen demand) in the wastewater. The major fungi genera that have been explored largely for heavy metals removal from aqueous solution are Rhizopus, Penicillium and Aspergillus.

2.Mechanisms involved in Bioremediation

Microorganisms in the bioremediation process convert organic contaminants to certain by-products like water and CO2 gas, or to other intermediates which they can further utilize for their growth. Microorganisms can deploy certain mechanisms such as dissolving metals, oxidizing or reducing transition metals, transformation, volatilizing and immobilization of heavy metals and bioleaching. Microbes can bring into effect certain defence mechanisms like formation of outer cell-membrane as a protective layer. The extracellular polymeric substances (EPS) present in the bacterial cell walls have been reported to show prominent actions on adsorption of heavy metals. EPS have been shown to be effective towards complexing heavy metals via exchange of H+ ions and metallic precipitation. Bioremediation research still needs to make considerable effort towards exploration of genetics and genome characteristics of various microorganisms for adsorption of heavy metals, kinetics studies and their metabolism

3.Biological Processes Involved in Bioremediation

Three major mechanisms which explain the ways by which microbes remove heavy metals ions from aqueous solution are as follows:

• Absorption of heavy metal ions by microorganisms on their cell surfaces.
• Uptake of heavy metal ions by microorganisms intra-cellular.
• Conversion of toxic form of heavy metals ions into non-toxic form by microorganisms.

Yun et al. (2001) studied various microorganisms for bioremediation and concluded that algae, particularly marine algae are amongst other organisms that are best for metal uptake.

3.1         Physical-Biological-Chemical Mechanism of Bioremediation

In the process of biosorption, a biosorbent must have high affinity for a sorbate (heavy metal ions), until equilibrium condition is reached between the biosorbent and sorbate. Saccharomyces cerevisiae deploys ion exchange mechanism and acts as a biosorbent to remove Zn (II) and Cd (II). The textile wastewater contains toxic heavy metals those can be absorbed effectively by Cunninghamella elegans. The efficient absorption and transformation of heavy metal ions can also be brought about by several fungi species such as Stachybotrys sp., Allescheriella sp., Botryosphaeria rhodina, Phlebia sp., Pleurotus pulmonarius and Botryosphaeria rhodina. Aspergillus parasitica and Cephalosporium aphidicola Klebsiella oxytoca can biodegrade soils contaminated with Pb (II) via biosorption process. Some species of fungi are resistant to mercury such as Neocosmospora vasinfecta, Verticillum terrestre, Hymenoscyphus ericae, etc. having ability to convert mercury (II) to a nontoxic oxidation state of mercury. Microbes are also known to secrete some bio-surfactants which form complexes and strong ionic bond interactions with metals. Later on, because of less interfacial tension, these formed complexes are desorbed from soil matrix to aqueous phase. Microorganisms can bio-remediate heavy metals either by aerobic oxidation or anaerobic oxidation. The aerobic oxidation occurs in the presence of abundant oxygen. The aerobic reactions are catalysed by various enzymes like hydroxylases, monooxygenases, dioxygenases, and oxidative dehalogenases. Peroxidases and lignases enzymes generate chemically reactive oxygen atoms which performs aerobic oxidation of heavy metals. Two consecutive steps occur in anaerobic processes. The activation reactions occur in first step. In second step anoxic acceptors of electrons carry out oxidative catabolism. The process of reducing the mobility of heavy metals collected from contaminated sites can be achieved by altering its chemistry and physiological nature is known as immobilization. Microbes use methylation (addition of methyl group), leaching, chelation and oxidation-reduction transformation of hazardous metals to mobilize heavy metals from contaminated sites. The heavy metals cannot be destructed completely but they can be converted into less hazardous, hydrophilic and precipitated form by the process of transformation. Heavy metals can be used by microorganisms as terminal electron acceptors. Detoxification process can also be used by microorganisms in which they reduce heavy metals and convert them into less hazardous form. This detoxification mechanism can be applied to eliminate heavy metals from the contaminated site. The two major means by which bacteria can build up resistance mechanism are detoxification and pumping out toxic metals from cells actively. In the detoxification process, toxic heavy metal state is transformed into less toxic form which ultimately converts it into unavailable form. The simultaneous oxidation and reduction of heavy metal ions occurs in the soil with the help of microorganisms. In this redox reaction, heavy metals are oxidised by microorganisms due to which heavy metals lose electrons. These released electrons are accepted by alternative acceptors of electrons such as ferric oxides, sulphates and nitrates. Microorganisms oxidize organic compounds having Fe (III) or Mn (IV) and derive energy. If Fe (III) is present abundantly, then there occurs a stimulation of anaerobic decomposition of organic contaminants. Biodegradation of chlorines occur via reductive de-chlorination, whereas electron acceptors are chlorinated solvents in respiration. The soluble state of Uranium (U6+) is converted into insoluble state (U4+) by Geobactor species. The toxic metals develop stress which can be reduced by various defense mechanisms developed by microorganisms. These are formation of complex, formation of binding proteins and peptides, exclusion and compartments formation (compartmentalization). The accumulation of heavy metals by microbes could be understood through expressing proteins that binds to metal and peptides such as metallothionein, phytochelatins, etc. The smtA gene expression as well as generating protein that binds to metal has been shown in Synechococcus sp. (cyanobacteria species). The expression of metallothionein present in the mouse on the cell surface and reduction of detrimental effects of cadmium (II) in the contaminated environment is achieved by genetically modified Ralstonia eutropha. Escherichia coli control the amount of accumulation of cadmium by expressing various proteins and peptides. The ten times increase in phytochelatins (PC) occurs due to simultaneous expression of both precursor glutathione (GSH) and phytochelatins (PC). This results in two times increase in cadmium accumulation. Metalloregulatory protein regulates the pathways of inherent resistant of heavy metals (arsenic, mercury, etc.) inside microbes. Bacteria acquire the property of heavy metals’ chelation, thus eliminating heavy metals by secreting exopolysaccharides from the contaminated environment. These exopolysaccharides have been reported in marine bacterium Enterobacter cloaceae. This bacterium can chelate up to 20% copper, 8% cobalt and 65% of cadmium at metal concentration of 100mg/L. Rhodobium marinum and Rhodobacter sphaeroides are purple nonsulfur bacterial isolates, reported to have the capacity of biosorption or biotransformation thereby eliminating ions of heavy metals such as lead, zinc, copper, cadmium, etc. from the contaminated environment.

3.2         Molecular Aspects of Bioremediation Phenomenon

Deinococcus geothermalis, a genetically engineered bacterium has been reported to reduce Hg because of mer operon expression in E.coli which codes for reduction of mercury. A bacterium that has resistance against mercury i.e., Cupriavidus metallidurans, a MSR33 strain was genetically altered through the introduction of a pTP6 palsmid. The merB and merG genes were provided through this plasmid. The main function of these genes i.e., merB and merG was to regulate biodegradation of mercury. These genes simultaneously synthesize mercuric reductase (MerA) and organomercurial lyase protein (MerB). Klebsiella pneumonia M426 bacteria degrade Hg by two different mechanisms. The first mechanism is Hg volatilisation by reducing Hg (II) to Hg (0). The second mechanism is Hg precipitation in which Hg precipitates out as an insoluble Hg due to the presence of volatile thiol (H2S). The plasmid namely pMR68 with mer novel genes were used to modify Pseudomonas strain to make it mercury resistant. Deinococcus radiodurans (radiation resistant bacterium) was genetically engineered to degrade toluene completely by using cloned genes of Pseudomonas putida of tod and xyl operons. Coenzymes and siderophores that are metal bounded are the main microbial metabolites involved in the degradation pathway. The various genes that are catabolic in nature are reported in bacteria. These catabolic genes are applied in bioremediation.

3.3   Hyper accumulators to Heavy Metal Tolerance

An elevated metal accumulation mechanism in plants without altering their metabolic mechanism has been studied in detail. The most common mechanism is the release of a complex organic compounds mixture through roots. The excreted substances contain compounds that are associated with metals, like siderophores. The iron absorption by plant organs is associated with siderophores. There is possibility of changes in these compounds under bioavailability of metals by plants (Maywald and Weigel, 1997). Malic acid and citric acid are the two most reported organic acids to capture metals. Delhouse and Ryan (1995) reported that aluminium exposition in wheat causes malic acid secretion from radicle apex, thus elevating resistance against metal. It was found that acids particularly organic in nature that were excluded from radicle of Arabidopsis were resistant for aluminium (Larsen et al.1998). Li et al. (2000) explained the function of mucilage in protection of roots from aluminium in Zea mays. Role of phytochelatins in physiological mechanisms of plants’ responses that were exposed to heavy metals with their capacity to bind metals is well published (Loeffler 1989, Mehra et al. 1996, Rauser 1999, Maitani et al. 1996).

9.Heavy Metals Tolerance of Plants through Biochemical Pathways

Bio-indicators are defined as those plants that possess the potential to colonize in the environments having elevated concentration of heavy metals thus, assimilate these heavy metals in their cells. These plants could be applied in the studies of phytoremediation in the contaminated soil laden from heavy metals. Biochemical changes are induced in plants due to heavy metals exposure. The plants those are sensitive to ions of heavy metals, there occurs an inhibition of enzymes involved in photosynthetic reactions. In heavy metal resistant plants, responses consist of the induction of peroxidase and enzymes involved in intermediate metabolism, stimulating tolerance to metallic stress. High assimilation of heavy metals in plants is an eco-physiological adaptation of the plants in heavy metal environments. To develop techniques in phytoremediation for soil contaminated with heavy metals, we can replicate the tolerance of plants to high metal contents. Metallothionein is a complex biochemical compounds called metal-proteins. Metallothionein participates in the metabolic pathways of plants exposed to ions of heavy metals. These metal-proteins are capable of making linkages with metals. Other proteins capable of linking metals are used for the storage of metals, as carriers, and proteins integrated in the ionic trans membranous channels. A short account on the role and chemical composition of the proteins engaged in heavy metal tolerance mechanisms is described as follows:

4.1  Phytochelatins (PC)

The role of phytochelatins in plant growth was explained by Kinnersely in 1993. The chemistry of phytochelatins and related proteins was described by Rauserin in 1995. The phytochelatins has a low molecular weight. Cd, Hg, Zn, Cu and Pb can be accumulated, detoxified and metabolized by these phytochelatins. Phytochelatins are almost absent in animals. The chemical constitution of PC is: (a-glutamilcystein) n-glycine, or (a-Glu-Cys) n-Gly, where n indicates two to eleven repetitive units, commonly 2 to 5 units are observed. These compounds are classified in metallothionein Class III and are synthesized enzymatically, unlike metallothionein Class I and II, those are genetically codified. In the family of Poaceae (maize) and in the order of Fables (bean), PC contains a-alanine serine or glutamic acid in its terminal end of glycine. This type of FQ is known as iso-PC. PCs can be induced by exposing autotrophic organisms, algae, monocotyledons, gymnosperms and also many species of fungi, to any heavy metals. Cadmium is a better activator, followed by Bi, Pb, Ag, Cu, Zn, Au and Hg. It has been observed that the biosynthesis of PC proceeds after metallic activation of an enzymatic constituent which utilize glutathion as substrate. This enzyme is glutamicysteine transpeptidase, commonly known as PC synthetase. The enzyme partially purified in vitro is functional only in the metallic ions presence. The activity of phytochelatin synthatase is studied in vegetable systems in vitro. It was observed that the enzyme was active up to the stage in which the quantity of metal was chelated completely by PC or by adding chelating agent like EDTA in the medium.

A mutant of Arabidopsis thaliana deficient in PC synthetase has expressed the vital function of PC in tolerance of heavy metals. The original variety of Arabidopsis thaliana is a Cd tolerant. On the other hand, the over exposition of the gene responsible for the enzyme of glutation synthetase of E.Coli implanted in Indian mustard plants enhanced remarkable tolerance to Cd in this species. This increased tolerance was due to an increase in the production of PCs.

4.2   Metallothioneins

It has low molecular weight and rich in cysteine. It constitutes the class of proteins and peptides associated with metals. The regulation of supply of essential nutrients, such as, copper and zinc is performed by metallothioneins. They are also responsible for protecting cell against toxic effects of the elevated concentrations of mercury, lead, cadmium, zinc, copper, etc. The metallothioneins are classified into three categories. Category I contain all the metallothionin whose residues of cysteine are localized in positions similar to that presented in corner meta-thionine and other mammiferos. Category II is protein-metallothionein which has no resemblance with the category I. Category III are polypeptides, atypical compounds of glutamilcysteine units. The drawback of this classification is that it does not determine the structural similarity. Therefore, a new classification has been proposed that will take into account the structural similarity with respect to the composition of amino acids, the length of the sequence of residues, positions of groups of cysteine and active sites as well as phylogenetic relations.

4.3         Biosynthesis and Regulation of FQ Synthetase

To explain the regulation of the enzymatic activity of PC synthetase, two fundamental models have been proposed. The first model describes that the N-terminal domain possesses the active site. This active site catalyzes the action of PC through its activation by the heavy metal interaction with the residues of cysteine and histidine. Severe mutation of the C-terminal region does not destroy the enzymatic activity. It is because the PC synthetase is expressed continuously and the levels of enzyme and mRNA are not changed on the exposure with heavy metals. This finding suggests that there exists no regulation at the transcriptional level.

4.4        Role of Chelate in Phytoextraction

Pb and Cd phytoextraction from a superficial soil by maize and mustard have been reported by Bricker et. al., (2001). The citrate and EDTA were most feasible in promoting Cd uptake by both maize and mustard. The heavy metals’ ions extraction from soils through plant is most effective in the chelate-enhanced phytoremediation. Chelators are added into soil to increase uptake of metals with the help of non-hyper accumulator plants. If heavy metals are not actively up-taken by plants from the contaminated soil, then they can leach into the ground and can pose the risk of ground water contamination. On the other hand, hyperaccumulator plants absorb, translocate the heavy metals through tonoplasts and assimilate these metals in the vacuoles. This leads to protecting cell activity from heavy metal toxicity. The two important determining factors responsible for developing phytoextraction techniques are as follows:

1) Appropriate selection of plant species

2) Rhizophere manipulation to accelerate metal uptake

Thlaspi goesingense (Brassicacae), a low biomass hyperaccumulator and Amaranthus hybridis, a high biomass that is not accumulating heavy metals were studied to find out the influence of ammonium sulphate and EDTA on the heavy metals’ assimilation into shoots. The findings of the study were that an application of 1g EDTA (Kg soil) increased considerably metal extractability with 1m NH4 SO4, whereas if we are applying only NH4 SO4, it would be effective. The application of EDTA enhanced the concentration of heavy metals in both of the plants. Heavy metals ions’ lability has increased largely in the soil treated with EDTA. Moreover, this ability remained for many weeks after the chelating agent was applied (Puschenreiter et al. 2001).

The synthetic apatite and EDTA in the brassica rapa was applied to change the bioavailability in contaminated soil for heavy metals. EDTA enhanced heavy metals translocation to the green organs of the plant from the roots. Apatite amendment reduces concentration of Pb in the leaves and lead, cadmium and zinc concentrations in the roots. EDTA application to apatite-amended soil considerably enhanced lead content in leaves. Further EDTA addition enhanced the share of bioavailability of Pb (Grcman et al.2001).The enhancement by chelate is plant and metal specific. Heavy metals accumulation, their phytoextraction, toxicity and leaching was increased by application of EDTA. (Grcman et al. 2001 a,b).

4.4.1    Protective Action of Phytochelatin (PC)

The plant vacuole is a chamber of transitory storage of peptides. In these plant vacuoles, peptides are dissociated, liberating, therefore, the metal in the vacuolar space, while the dissociated peptide is disintegrated. Through this means, the intracellular heavy metal gives protection to the enzymes sensitive to metals. Ortiz et al. in 1992 and 1995 found out the possibility to determine the presence of one gene, HMT1 in mutant stock of S. pombe. This HMT1 gene codifies one protein localized in the vacuolar membrane or tonoplast. The transportation of PC-Cd complex to the interior of the vacuole is performed by HMT1 gene. Vogeli- Lange and Wagner in 1990 studied the transport of PC up to the vacuole in the mesophyll cells of tobacco exposed to Cd. In this location, there is also a presence of most of the metal and enzyme that is confined to the interior of vacuole, thus protecting cell metabolism from metal toxicity.

10.Tissue Culture Techniques are Applied in the Process of Bioremediation

Somatic hybridisation is a technique by which trait transfer for heavy metal hyperaccumulation is possible. It was demonstrated by Brewer et al.in 1999 that hybrids of the zinc hyperaccumulator Brassica napus and T. caerulescens were chosen for tolerance of zinc. They were then grown until flowering in Zn enriched soils. The accumulated levels of Zn and Cd were toxic for Brassica napus. Cr and Ni tolerance in Echinochloa colona was developed by Samantaray et al. in 2001 through callus culture and plant regeneration in both tolerant and non-tolerant callii. In comparison to the susceptible plant, the regenerated hybrid showed tolerance to heavy metal. In metal exclusion and metal uptake, several protein-binded transporters play a crucial role.

Concluding remarks

The heavy metal ions bio-sorption on microbial surface, their intracellular uptake by microorganisms and chemical transformation of heavy metal ions by microbes are the three major biological phenomena involved in bioremediation. Microorganisms perform detoxification process in which trace elements and ions of heavy metals act as terminal acceptors of electrons. These heavy metal ions in turn are reduced into non-toxic forms by microorganisms. Microorganisms use certain mechanisms such as dissolving metals, oxidising or reducing transition metals, transformation, volatilizing and immobilization of heavy metals and bioleaching to remediate various toxic heavy metals. Microbes can bring into effect certain defence mechanisms. These defence mechanisms can be outer cell membrane formation and pumps for efflux of solvents. The function of this outer cell membrane is to give protection to microbes against detrimental effects of heavy metals. The nature of outer membrane is usually lipophilic. Lastly, application of bioremediation technology in remediation of heavy metals still requires a lot of studies on exploring concepts of genetics, gene regulation and characteristics of genome of various microorganisms applied in adsorption of heavy metals, their kinetics and metabolism.