1

BIOREMEDIATION OF METAL

CONTAMINATED SOIL

BY: HIMANSHU ARORAROLLNO. 03M.SC (F) EVS

2Contents Introduction Sources of Metals in the Soil Principles of Bioremediation Types of Bioremediation Microorganisms Used in

Bioremediation Mechanisms of Bioremediation

Biosorption Biimmobolization Bioleaching Biomineralization

3 Phytoremediation Hyperaccumulators Plant Microbe Interaction: Rhizoremediation Designer Microbe Approach Advantages and Disadvantages of Bioremediation Case Study Conclusions References

4Introduction Indiscriminate release of metals into the soil is a major

health concern worldwide, as they cannot be broken down to non-toxic forms and therefore have long-lasting effects on the ecosystem. Many of them are toxic even at very low concentrations; As, Cd, Cu, Pb, Hg, Ni, Ag, Zn etc. are not only cytotoxic but also carcinogenic and mutagenic in nature.

There are several techniques to remove these metals, including chemical precipitation, oxidation or reduction, filtration, ion-exchange, reverse osmosis, membrane technology, evaporation and electrochemical treatment. But most of these techniques become ineffective when the concentrations of heavy metals are less than 100 mg/L

5 Alternatively, use of microorganisms and plants

for remediation purposes is thus a possible solution for metal pollution since it includes sustainable remediation technologies to rectify and re-establish the natural condition of soil.

Bioremediation is the collective range of clean up methods by using natural microorganism (such as bacteria, Fungi, biopolymers) and plants (phytoremediation) to degrade hazardous organic contaminants or convert hazardous inorganic contaminants to environmentally less toxic or nontoxic compounds of safe levels in soils, subsurface materials, water, sludges, and residues.

6Sources of Metals in the Soil Metals occur naturally in the environment from

pedogenetic processes of weathering of parent materials and also through anthropogenic sources.

Metals

Anthropogenic Sources1. As: Pesticides, wood preservatives, biosolids, ore mining and smelting 2. Cd: Paints and pigments, plastic stabilizers, electroplating, phosphate fertilizers 3. Cr: Tanneries, steel industries, fly ash 4. Cu: Pesticides, fertilizers, biosolids, ore mining and smelting 5. Hg: Au-Ag mining, coal combustion, medical waste 6. Ni: Effluent, kitchen appliances, surgical instruments, automobile batteries 7. Pb: Aerial emission from combustion of leaded fuel, batteries waste, insecticide and herbicides

Natural Sources

1. Weathering of minerals 2. Erosion and volcanic activities 3. Forest fires and biogenic source 4. Particles released by vegetation

7Principles of Bioremediation Bioremediation is based on the idea that organisms are

capable to take in pollutants from the environment and use them to enhance their growth and metabolism. Or convert them from a toxic to a nontoxic or less toxic.

Bacteria and fungi are well known for degrading complex molecules and transform the product into part of their metabolism

8Types of Bioremediation On the basis of removal and transportation of wastes

for treatment there are basically two methods. These are 1) in-situ bioremediation and 2) ex-situ bioremediation.

1) In-situ Bioremediation No need to excavate or remove soils or water in order to

accomplish remediation. In-situ biodegradation involves supplying oxygen and nutrients

by circulating aqueous solutions through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants.

It is of two types: Intrinsic bioremediation and Engineered In-situ bioremediation.

9 In-situ bioremediation approach deals with stimulation of

indigenous or naturally occurring microbial populations by feeding them nutrients and oxygen to increase their metabolic activity.

Whereas engineered bioremediation approach involves the introduction of certain microorganisms to the site of contamination.

There are different techniques of in-situ bioremediation: Biosparging, Bioaugumentation, Bioventing.• Biosparging involves the injection of air under pressure below the

water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria.

• Bioaugumentation involves practice of adding specialized microbes or their enzyme preparation to the polluted sites to accumulate transformation or stabilization of specific pollutants.

• Bioventing involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria..

102) Ex-situ Bioremediation Ex-situ bioremediation techniques involve the

excavation or removal of contaminated soil from ground.

Depending on the state of the contaminant to be removed, ex-situ bioremediation is classified as a) solid phase system and b) slurry phase systems. a) Solid Phase System The Solid phase treatment includes wastes such as

leaves, animal manures and agricultural wastes and problematic wastes like domestic and industrial wastes, sewage sludge and municipal solid wastes.

Solid phase soil treatment processes include land farming, soil biopiles, and composting.

11 Land farming is a simple technique in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded. The goal is to stimulate indigenous biodegradative microorganisms and facilitate their aerobic degradation of contaminants.

Composting is a technique that involves combining contaminated soil with nonhazardous organic amendants such as manure or agricultural wastes. The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting.

Biopiles are a hybrid of land farming and composting. Essentially, engineered cells are constructed as aerated composted piles. Typically used for treatment of surface contamination with petroleum hydrocarbons they are a refined version of land farming that end to control physical losses of the contaminants by leaching and volatilization. Biopiles provide a favorable environment for indigenous aerobic and anaerobic microorganisms

12b) Slurry Phase System Contaminated soil is combined with water and other

additives in a large tank called a bioreactor and mixed to keep the micro organisms, which are already present in the soil, in contact with the contaminants in the soil.

Nutrients and oxygen are added and conditions in the bioreactor are controlled to create the optimum environment for the microorganisms to degrade the contaminants.

When the treatment is completed, the water is removed from the solids, which are disposed of or treated further if they still contain pollutants.

13Microorganisms Used in Bioremediation There are a number of microorganisms that can be used to

remove metal from environment, such as bacteria, fungi, yeast and algae.

Because of the adaptability of microbes and other biological systems, these can be used to degrade or remediate environmental hazards.

The organisms that are utilized vary, depending on the chemical nature of the polluting agents, and are to be selected carefully as they only survive within a limited range of chemical contaminants.

The microorganisms can be subdivided into following groups:I. Aerobic: Degrade metals in presence of Oxygen.II. Anaerobic: Degrade metals in absence of Oxygen.III. Methylotrophs: Aerobic bacteria that grow utilizing methane for

carbon and energy.

14IV. Lignolytic Fungi: The ability of white-rot fungi (kind of Lignolytic Fungi) to adsorb and accumulate metals together with the excellent mechanical properties of fungal mycelial provide an opportunity for application of fungal mycelia in selective sorption of individual heavy metal ions.

Microorganisms ElementsBacillus spp. Pseudomonas aeruginosa

Cu, Zn

Zooglea spp. Citrobacter spp.

U, Cu, NiCo, Ni, Cd

Citrobacter spp. Chlorella vulgaris

Cd, U, PbAu, Cu, Ni, U, Pb, Hg, Zn

Aspergilus niger Cd, Zn, Ag, Th, UPleurotus ostreatus Cd, Cu, ZnRhizopus arrhizus Ag, Hg, P, Cd, Pb, CaStereum hirsutum Cd, Co, Cu, NiPhormidium valderium Cd, PbGanoderma applantus Cu, Hg, Pb

Microbes Utilize the Metals

15Mechanisms of Bioremediation Remediation of environment niches such as soil,

sediments and water amended with metals can be achieved through biologically encoded changes in the oxidation state.

Microorganisms are omnipresent that dominate in metal-contaminated soil and can easily convert metals into non-toxic forms but microbes are unable to simplify them into harmless compounds, they should be used according to their specialization for the type of contaminants.

Microorganisms are capable of two-way defense viz. production of degradative enzymes for the target pollutants as well as resistance to relevant heavy metals.

Metal-microbe interactions like biosorption and bioaccumulation, biomineralization, bioleaching and bioimmobilization (enzyme-catalyzed transformations) are used as the mechanisms for the removal of metals from the soil.

16Biosorption It involves a solid phase(sorbent or biosorbent;

Biological material) and a liquid phase (solvent, normally water) containing a dissolved species to be sorbed (sorbate, metal ions). Due to higher affinity of the sorbent for the sorbate species, the later is attracted and removed by different mechanisms.

The process continue until the equilibrium is established between the amount of solid-bound sorbate species and its portion remaining in the solution.

The metal biosorption process by living cells is a two-step process: 1) Passive biosorption, 2) Active biosorption.

In Passive biosorption, metal ions are adsorbed to the surface of cells by interactions between metals and functional groups displayed on the surface of cells. Difference in the cell wall composition of different microbial groups, cause significant difference in the type and amount of metal ion binding to them. E.g. In E. coli K12, peptidoglycan was found to be a potent binder and carboxylate groups were the principal component in metal binding.

17 Passive biosorption is metabolically independent and proceeds rapidly

by any one or a combination of the metal binding mechanism: Coordination, complexation, ion exchange, electrostatic interaction, metal precipitation, physical adsorption or extracellular interaction

In active biosorption, metal ions penetrate the cell membrane and enters into the cell.

E.g. Uptake of Pb by dried mass of Chlorella vulgaris, an alga. E.g. Saccharomyces cerevisiae acts as a biosorbent for the removal of

Zn (II) and Cd (II) through the ion exchange mechanism Bioaccumulation involves metal binding on intracellular

compounds, metal binding proteins, intracellular precipitation, methylation and other mechanisms. The uptake process usually involves adsorption of heavy metal ions at the bacterial cell wall or cell membrane through interactions with various functional groups and/or transport into the cell with subsequent transformation.

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19Bioimmobilization The mechanism used to reduce the mobilization of heavy metals

from contaminated sites by changing the physical or chemical state of the toxic metals is called immobilization.

It involves aerobic and anaerobic activity of the microorganisms Aerobic degradation or oxidative precipitation often involves

introduction of oxygen atoms into the reactions mediated by monooxygenases, dioxygenases, hydroxylases, oxidative dehalogenases, or chemically reactive oxygen atoms generated by enzymes such as ligninases or peroxidases.

Anaerobic degradations or reductive precipitation of contaminants involve initial activation reactions followed by oxidative catabolism mediated by anoxic electron acceptors.

This process is also called as enzyme catalyzed biotransformation.

20

e- oxidation

metal reducing bacterium

Fe(III),SO4-

Fe(II),H2S

Red Oxi Red

Cr(III)insoluble,immobile less toxic

Cr(VI)solublemobiletoxic

Microbes precipitate the metals by changing their valence

21Bioleaching Microbes mobilize the heavy metals from the

contaminated sites by leaching, chelation, methylation and redox transformation of toxic metals.

Heavy metals can never be destroyed completely, but the process transforms their oxidation state or organic complex, so that they become water-soluble, less toxic and precipitated

Bioleaching facilitates cycling of metals by a process close to natural biogeochemical cycles, reducing the demand for resources such as ores. E.g. Chemolithotrophic bacteria, mainly

Acidothiobacillus ferrooxidans and A. thiooxidans are widely used in the process of bioleaching. Oxidation of S generates sulfuric acid (decrease soil pH) and production of organic acids or sulfuric acid which form metal chelates.

22Biomineralization It is the formation of minerals by complex processes directly

or indirectly driven by living organisms, associated with metabolic activity due to the impact of compound produced in the intracellular or extracellular matrix of the organism.

Microbes provide ions for crystal formation and determine size, shape, and structure, as well as physical characteristics of the biominerals.

Almost all groups of minerals, including carbonates, phosphates, sulfates and sulfides, arsenates, silica, chlorides, fluorides, oxides, hydroxides and Fe-Mn-oxides are known to be produced by biomineralization

Biomineralizaton of Struvite (MgNH4PO4 . 6H2O) by strains of Bacillus, Corynebacterium, Kurthia, Staphylococcus generas.

23Phytoremediation

24Hyperaccumulators Exhibit higher heavy metal tolerance and accumulating

abilities compared to other plants. Many such plants like Arabidopsis halleri and Solanum

nigrum L. have been utilized for phytoremediation of cadmium. Metal Plant Species

Cd, Cu, Pb, Zn Salix spp. (Salix viminalis, Salix fragilis)

Cd Castor (Ricinus communis)Cd, Pb, Zn Corn (Zea mays) Cd, Cu, Pb, Zn Populus spp. (Populus

deltoides, Populus nigra, Populus trichocarpa)

Cd, Cu, Ni, Pb Jatropha (Jatropha curcas L.)Hg Populus deltoidesSe Brassica juncea, Astragalus

bisulcatus Zn Populus canescens

25Plant Microbe Interaction: Rhizoremediation Plants combined to some microorganisms to increase the

efficiency of contaminants extraction; such technique is called rhizoremediation.

In the rhizospheric degradation process, the metal toxicity to plants can be reduced by the use of plant growth-promoting bacteria, free-living soil microorganisms that exert beneficial effects on plant growth.

In this process, plants can stimulate microbial activity about 10–100 times by the secretion of exudates which contain carbohydrates, amino acids, flavonoids etc.

E.g. Nickel-resistant soil bacterium Kluyvera ascorbata SUD 165 promoted the growth of Brassica campestris in the presence of high concentration of nickel due to its ability to lower the level of ethylene stress in the seedlings.

26Designer Microbe Approach Genetically engineered microorganisms (GEM) are

organisms whose genetic material has been altered using recombinant DNA technology to generate a character-specific efficient strain for bioremediation of soil, water and activated sludge by exhibiting enhanced degrading capabilities against a wide range of chemical contaminants.

Offers the advantage of constructing microbial strains which can withstand adverse stressful situations and can be used as a bioremediators under various and complex environmental conditions.

E.g. Bacteria like Escherichia coli and Moreaxella sp. expressing phytochelatin 20 on the cell surface have been shown to accumulate 25 times more Cd or Hg than the wild-type strains

27Heavy Metal GEM Expressed Gene

As E. coli strain Metalloregulatory protein ArsR

Cd2+ E .coli strain SpPCS

Cr6+ Methylococcus capsulatus

CrR

Cr P. putida strain Chromate reductase (ChrR)

Cd2+, Hg Ralstonia eutropha CH34, Deinococcus radiodurans

merA Limitations Chances of sustainability of the recombinant bacteria

population in soil, with various environmental conditions and competition from native bacterial populations.

The molecular approaches have been applied to only limited bacterial strains like Escherichia coli, Pseudomonas putida, Bacillus subtilis etc.

28Advantages and Disadvantages of BioremediationAdvantages

Minimal exposure of on site workers to the contaminant

Long term protection of public health

The Cheapest of all methods of pollutant removal

The process can be done on site with a minimum amount of space and equipment

Eliminates the need to transport of hazardous material

Uses natural process Perform the degradation in an

acceptable time frame

Disadvantages Cost overrun Failure to meet targets Poor management Climate Issue Regulatory compliance concern Release of contaminants to

environment It can takes a few month to as

long as a few years. Not all organic compounds are

biodegradable There are some concerns that the

products of biodegradation may be more toxic than it’s parental form

29Case Study BIOREMEDIATION OF SOIL BY REMOVING HEAVY

METALS USING Saccharomyces cerevisiae by D. Damodaran, G. Suresh, Raja Mohan B. presented in 2nd International Conference on Environmental Science and Technology in 2011.

In the industrial complexes of Mangalore there are nearly 1039 industries ranging from small scale to large scale industries, out of which 156 of metal processing industries are present.

Soil samples were collected from potential sites of metal contamination near areas of Kudremukh Iron Ore Company Ltd (KIOCL), Gurupur River, lay bye areas of NH-17 and Bikampady Industrial estate areas.

Saccharomyces cerevisiae used in the study was obtained from National Chemical Laboratory, Pune, India.

In this study Saccharomyces cerevisiae was used for the removal of heavy metals like Lead and Cadmium from contaminated soil.

30 YPAD medium was prepared and amended with various amounts of heavy metals viz., 3CdSO4.8H2O and Pb (NO3)2 to achieve the desired concentration of 5, 10, 25, 50, 100, 150, 200 and 250 ppm for Pb2+ and 30, 75, 150, 225, 300,350 and 400 ppm for Cd2

The tolerance of Saccharomyces cerevisiae against the metals was found to be upto 250 ppm and for Pb2+, 500 ppm for Cd2+.

The results revealed that biosorption of about 67-82% of Pd2+ and 73-79 % of Cd2+ was attained within 30 days. The time taken for maximum sorption of Pb2+ and Cd2+ was 30 days for soil containing 100 and 300 ppm of Pb2+and Cd2+ respectively.

31Conclusions Bioremediation provides a technique for cleaning up

pollution by enhancing the natural biodegradation processes. So by developing an understanding of microbial communities

and their response to the natural environment and pollutants, expanding the knowledge of the genetics of the microbes to increase capabilities to degrade pollutants, conducting field trials of new bioremediation techniques which are cost effective, and dedicating sites which are set aside for long term research purpose, these opportunities offer potential for significant advances.

Phytoremediation methods are well suited for use at very large field sites where other methods of remediation are not cost effective or practicable.

32References Baldrian, Petr. "Interactions of Heavy Metals with White-

rot Fungi." Enzyme and Microbial Technology 32.1 (2003): 78-91. Web.

Dixit, R., Wasiullah, E., Malaviya, D., Pandiyan, K., Singh, U., Sahu, A., . . . Paul, D. (2015). Bioremediation of Heavy Metals from Soil and Aquatic Environment: An Overview of Principles and Criteria of Fundamental Processes. Sustainability,7(2), 2189-2212

Nareshkumar, R., Nagendran, R., & Parvathi, K. (2007, August). Microbial solubilization of heavy metals from soils using indigenous sulfer oxidizing bacterium: Effects of sulfer/soil ratio. Journal of Scientific and Industrial Research, 66, 680-683.

Girma, G. (2015). MICROBIAL BIOREMEDIATION OF SOME HEAVY METALS IN SOILS : AN UPDATED REVIEW. Ind.J.Sci.Res., 6(1), 147-161.

Kothe, E., & Varma, A. (2012). Bio-geo interactions in metal-contaminated soils. Heidelberg: Springer.