Bioremediation Techniques (Methods) PDF

Introduction: “Remediate” means to solve a problem and “bioremediate” means to use biological organisms to solve an environmental problem such as contaminated soil or ground water. Bioremediation specially deals with restoration of environment already contaminated. It offers a cost- ef...

Introduction: “Remediate” means to solve a problem and “bioremediate” means to use biological organisms to solve an environmental problem such as contaminated soil or ground water. Bioremediation specially deals with restoration of environment already contaminated. It offers a cost- effective, permanent solution to clean-up soils contaminated with xenobiotic compounds. The general components and characteristics of bioremediation constitute three important aspects: Microbial systems Type of contaminant Geological and chemical conditions at the contaminated site Definition Bioremediation is defined as the use of biological treatment systems to destroy, or reduce the concentration of hazardous waste from contaminated sites. The American Academy of Microbiology defined Bioremediation as the use of living organisms to reduce or eliminate environmental hazards resulting from accumulation of toxic chemicals and other hazardous wastes. Bioremediation can also be defined as the complete removal of pollutants and their toxicity through the metabolic reaction mediated by microorganisms. Bioremediation is the manipulation of living systems to bring about desired chemical and physical changes in the confined and regulated environment. Here is how it works: A: The waste material is examined and certain bacteria are isolated based on their efficiency at digesting and converting the waste. B: The bacteria then go through a series of tests for performance and safety. C: bacteria are next placed back in the waste environment in high concentrations. D: The bacteria grow and thrive and in the process digest and convert the waste into carbon dioxide and water. E: The bacteria die-off naturally. Basis of Bioremediation Bioremediation is based on microbial metabolism. Xenobiotics can serve as substrates for microbial growth and energy. The xenobiotic supports microbial growth if it is metabolized. The Xenobiotics becomes a sources of carbon, nitrogen, sulphur and energy. If the xenobiotic is added to soil, the microbial population increases. Microorganisms that specifically grow on the xenobiotic can be isolated by enrichment culture. Advantages and Disadvantages of Bioremediation Can be done on site. Minimum site disruption is caused. Eliminates transportation and long-term liabilities – cost effective technology. Uses biological system, often less expensive Can be coupled with other treatment techniques. Large volumes of soil may be treated. Likely to be supported by the public since it is viewed as a natural process. Disadvantages: Some chemical compounds are not biodegradable. Extensive monitoring is required. Each site has specific requirements. Potential production of toxic unknown sub-products is possible. Strong scientific support is needed. Complex wastes can inhibit biological activity. Principles/ Types of Bioremediation Bioremediation explores the genetic diversity and metabolic versatility of microbes for the transformation of contaminants in to less harmful end products. The selection of bioremediation system depends on the constituent of the contaminants and it involves a multidisciplinary approach requiring knowledge from individuals with expertise in chemistry, microbiology, geology, environmental engineering, chemical engineering, soil science etc. The Bioremediation treatment technologies can be categorized as follows. Biodegradation: where the breaking down of a compound or substance is achieved with living organisms such as bacteria or fungi. These could be indigenous to the area, or could be introduced. Biostimulation: Where the natural or introduced population of microbes in an area are enhanced through addition of nutrients, engineering or other manipulation of an area. This speeds up the natural remediation process. Bioaugmentation: Where specific living organisms are added to a site or material to achieve a desired bioremediation effect.  Biorestoration : Restoration to original or near original state using living microbes. Bioattenuation: A method of monitoring the natural program of degradation to ensure that contaminant concentration decreases with time at relevant sampling point. Bioventing: Treating contaminated soils by drawing oxygen through the soil to stimulate microbial growth and activity. It is needed when it becomes necessary to supply oxygen from air. Biomineralization(Biocrystallization): The process whereby microbially generated ligands or microbially mediated changes in the cellular microbial environment cause precipitation of heavy metals as biomass-bound crystalline deposits.  Acinetobactor calcoaceticus Bacteria used in Bioremediation: Acinetobactor calcoaceticus Arthrobactor/ Brevibacterium sp. Oceanospirillum sp. Pseudomonas putida Pseudomonas sp. Trichosporon sp. Alcaligenes sp Flavobactor/ Cytophaga sp. Pseudomonas fluorescens Pseudomonas stutzeri Pseudomonas putida Pseudomonas vesicularis Vibrio sp Nocardia sp Essential characteristic of microbes for bioremediation: Microbial degradation depends on several conditions which can be summarized as follows. Presence of microbes with the capacity to degrade the target compounds. Substrate being accessible to the organisms and capable of being used as energy and carbon sources. Presence of inducer to cause synthesis of specific enzymes for the target compounds. Presence of an appropriate electron acceptor- donor systems. Ideal moisture and pH for microbial growth. Nutrients for microbial growth and enzyme production. Optimum temperature to support microbial activity. Absence of toxic substance. Ideal conditions to minimize competitive organisms for those conducting designed reactions. Characterization of Essential factors for bioremediation All the contaminated sites are not suitable for treatment with bioremediation techniques. It is essential to know information about three closely related aspects : The chemical nature of contamination, The geohydrochemical properties, and The biodegradation potential for the site. Bioremediation Mechanisms: The chief ways by which remediation may be accomplished include Biosorption, Bioaccumulation, Precipitation, Reduction, Solubilization. Biosorption: Biosorption is the sequestering of chemicals( usually heavy metals) by materials of biological origin. Biosorption uses the extraordinary capacity of certain types of microbial and seaweed biomass to bind and concentrate metals. These bio-materials, are used just like "magic granules" which remove and concentrate heavy-metals from industrial effluents.  Rhizopus sp. Some types of biomass are waste byproducts of large-scale industrial fermentations (e.g., the mold Rhizopus or the bacterium Bacillus subtilis). Other metal-binding biomass types, such as certain abundant seaweeds (particularly brown algae, e.g., Sargassum, Ecklonia), can be readily harvested from the oceans. Threat from the Environment: It has been established that dissolved metals (particularly heavy metals) escaping into the environment pose a serious health hazard. They accumulate in living tissues throughout the food chain which has humans at its top, multiplying the danger. Thus, it is necessary to control emissions of heavy metals into the environment. Ranking of Risks Associated with Various Metals: High risk Moderate risk Low risk Cd Cr Al Pb Co Fe Hg Cu Ni Zn Physical adsorption: Physical adsorption takes place with the help of van der Waals' forces. Volesky 1988, hypothesized that uranium, cadmium, zinc, copper and cobalt biosorption by dead biomasses of algae, fungi and yeasts takes place through electrostatic interactions between the metal ions in solutions and cell walls of microbes. e.g.., Copper biosorption - Zoogloea ramigera and alga Chlorella vulgaris Ion Exchange: Cell walls of microorganisms contain polysaccharides and bivalent metal ions exchange with the counter ions of the polysaccharides The biosorption of copper by fungi Ganoderma lucidium and Aspergillus niger was also up taken by ion exchange mechanism Chelation : The word chelation is derived from the Greek word chele, which means claw, and is defined as the firm binding of a metal ion with an organic molecule (ligand) to form a ring structure. The resulting ring structure protects the mineral from entering into unwanted chemical reactions. Examples include the carbonate (CO32–) and oxalate (C2O42–) ions: Coordination (Complex Formation): The metal removal from solution may also take place by complex formation on the cell surface after the interaction between the metal and the active groups. Micro­organisms may also produce organic acids (e.g., citric, oxalic, gluonic, fumaric, lactic and malic acids), which may chelate toxic metals resulting in the formation of metallo-organic molecules. These organic acids help in the solubilisation of metal compounds and their leaching from their surfaces. Metals may be biosorbed or complexed by carboxyl groups found in microbial polysaccharides and other polymers. e.g., calcium, magnesium, cadmium, zinc, copper and mercury accumulation by Pseudomonas syringae. Organisms for Biosorption There is a wide variety of microorganisms including bacteria, fungi, yeast, and algae, that can interact with metals and radionuclides and transform them through several mechanisms. Examples of toxic heavy metals accumulating microorganisms: Organism Element Citrobacter sp. Lead, Cadmium Thiobacillus ferrooxidans Silver Bacillus cereus Cadmium Bacillus subtilis Chromium Pseudomonas aeruginosa Uranium Micrococcus luteus Strontium Rhizopus arrhizus Mercury Aspergillus Thorium Saccharomyces cerevisiae Uranium Advantages of Biosorption Biosorption is highly competitive with the presently available technologies like ion exchange, electrodialysis, reverse osmosis, etc. Some of the key features of biosorption compared to conventional processes include: competitive performance heavy metal selectivity cost-effectiveness regenerative no sludge generation. Bioaccumulation: Bioaccumulation refers to the accumulation of substances, such as pesticides, or other organic chemicals in an organism. Bioaccumulation occurs when an organism absorbs a toxic substance at a rate greater than that at which the substance is lost. Thus, the longer the biological half-life of the substance the greater the risk of chronic poisoning, even if environmental levels of the toxin are not very high. The concentration of a foreign substance within a biological system is dependent upon rate of uptake, duration of exposure and rate at which it is being eliminated or reached upon by the system.  The extent up to which a chemical shall be absorbed and bioaccumulated by an organism depends on its solubility in fats or its lipophilicity. Pollutants soluble in lipoid material are capable of forming complexes with macromolecules within the cell may be stored for long duration of time. Examples: Lipid (fat) soluble poisons include tetra-ethyl lead compounds (the lead in leaded petrol) and DDT. These compounds are stored in the body's fat, and when the fatty tissues are used for energy, the compounds are released and cause acute poisoning. Precipitation: Contaminants react with a product of microbial metabolism to yield water in soluble derivative. The removal of such precipitate constitute remediation. Sulphides and phosphates are the common precipitates formed in microbes due to the production of Hydrogen sulphide from sulphates and inorganic phosphates from organic phosphate compounds. The hydrogen sulphide / phosphate forms insoluble derivatives with a number of metallic ions. Microbial sulphate reduction for remediation is important in the use of constructed wet lands to remove metals from acids. Reductive halogenation : This is potentially important in the detoxification of halogenated organic compounds.  The halogen atom (such as chlorine) of the contaminant molecule is replaced by hydrogen atom due to catalytic reaction of microbes. Two electrons are added, and the contaminant become less toxic and susceptible to further microbial decay than the parent compounds. Reduction: Microbes can bring about the reduction of a wide array of inorganic anions and cations. Nitrate, sulphate and carbonate are the non-metallic anions that are reduced microbiologically. The reductions convert a higher oxidation state of the element to a lower one, e.g. Hg(II) to Hg(0), Fe(III) to Fe(II), Se(VI) to Se(0), Mn(IV) to Mn(II). The reduction changes the toxicity, water solubility and mobility of the element. Metabolic process involved in Bioremediation: Microbes that live virtually everywhere are the vital components for bioremediation. Microbial enzymes acts as catalysts in degradative (catabolic) reactions that provide energy and material for synthesis of additional microbial cells. In general, the biochemical process can be divided into two groups: Fermentation and Respiration. Two types of metabolism exist, depending on the type of electron acceptor. Respiration Fermentation Strategies for the improvement of Bioremediation Techniques: Addition of air or oxygen as the terminal electron acceptor for catabolic activity of microbes involved in bioremediation process. Since the oxygen supplied to the in situ process is limited, pump and treat option improves biodegradation. As the water solubility of alternative electron acceptors like nitrate, sulphate etc., are high, they have the potential to improve electron acceptor bioactivity. Composition of microbial communities improves bioactivity. Organisms that are sensitive to water activity maximize bioactivity Biomass immobilization and Bioremediation One of the major problems in the use of microorganisms for the biological treatment of waste waters is the recovery from treated effluents. Immobilization technique is the best solution to solve this problem. Cell immobilization can be defined as the confinement of whole cells in an insoluble phase, which permits the free exchange of solutes from and towards the biomass but at the same time isolates the cells from their surrounding medium. Hydrochloric, nitric, sulphuric acids are the eluents. Metals such as Au, Ag, and Hg can be desorbed using chelating compounds such as EDTA or complexing agents such as thiourea. Substances for immobilization A higher number of substances have been examined for the immobilization of different types of biomass. Polyacrylamide, calcium alginate and silica can be used as support for algal immobilization. Agar, agarose, K- carrageenan and diatomaceous earth are the natural materials used as immobilization matrices. Polyurethane and polyvinyl foams, polyacrylamide, ceramics, epoxy resin and glass beads are the synthetic materials frequently used as biosolvent supports. Cell immobilization Techniques Blanco et al., classified immobilization techniques as physical and chemical depending on the type of binding involved. Physical immobilization: is based on the natural tendency of cells to form flocs or to absorb onto inert surfaces. Chemical immobilization: Binding to supporting surface, cross linking and entrapment into the porous structure of a polymeric matrix are the important chemical immobilization strategies. Gelation, polymerization and insolubilization are the different methods of entrapment. Microbes could be immobilized either in a viable or non-viable form depending on its end use The immobilized cells have been investigated for their application : pharmaceutical food and dairy industries in waste water treatment in biofuels and in the synthesis of various chemicals.  List of immobilized microbes in bioremediation Organisms Support Application Pseudomonas putida Agar Degradation of caffeine Alcaligens denitrificans Alginate Degradation of n-valeric acid Nocardia rhodochrons alginate /polyacrylamide cholesterol Alcaligens sp. Polyacrylamide Divalent metal cation and Enterobacter and DDT citrobacter sp. Escherichia coli Carrageenan Aromatic rings (xenobiotics) BIOREMEDIATION TECHNIQUES (METHODS).. Techniques in Bioremediation: In situ remediation Ex situ remediation Bioventing Land farming Biostimulation Composting Bioaugmentation Biopiling Air sparging Bioreactors Techniques in Bioremediation Bioremediation can be broadly subdivided into in situ treatment and ex situ treatment. In situ treatment ( Contaminant treated at the same site = on site treatment) In situ bioremediation is the in place treatment of a contaminated site. Which directly involves the contact between microorganisms and the dissolved and sorbed contaminants. Bioventing: Aerobic biodegradation of soil contamination is stimulated by delivery of oxygen to the subsurface, by injecting or extracting air through soil at low flow rates or simply boring holes in the soil. This technology was designed primarily to treat soil contamination by fuel, non haloginated Volatile organic compounds (VOC) and semi volatile organic compounds (SVOC) , pesticides.  It involves supply of air to the soil to be treated, using a combination of pumps and blowers. Often bioventing is combined with another technique called soil vapour extraction for faster remediation of highly contaminated sites. In soil vapour extraction, much higher flow rates are used to extract volatile hydrocorbon and petroleum contaminated sites. Bioventing requires the presence of indigenous organisms capable of degrading the contaminants of interest. It is necessary that the contaminants be Merits : Bioventing uses simple, inexpensive, low-maintenance equipment that can be left unattended for long periods of time. Biostimulation : This involves stimulation of degradative activities of indigenous microbial in the subsurface by the addition of nutrients. These include adding a nitrogen source, a phosphorous source and a myriad of trace elements. This technology was designed primarily to treat soil contamination by fuel, non halogenated Volatile organic compounds (VOC) and semi volatile organic compounds (SVOC) , pesticides The cost of biostimulation technology vary tremendously from site to site, this technology tend to be among the cheapest of alternatives. There are numerous short comings with this treatment: we cant be certain that those organisms present are the most suitable to degrade all materials present in the contaminant. The organism stimulated can eliminate the primary substrates, but do not degrade the specially targeted sites. Bioaugmentation: If the indigenous population of microbes is not capable of fully degrading the contaminants, external source of super microbes are applied to the contaminated media along with the nutrients. These externally added microbes multiply and increase in population while degrading the contaminants, using the added nutrients. The selected organism must de carefully matched to the waste contamination present in the soil , as well as the metabolites formed. They must favorably compete with the ubiquitous organisms found in the expected environmental condition. By selecting the microbial consortium before hand, it is possible to select organisms that will not produce nuisance odours such as hydrogen sulphide. Air Sparging : Air Sparging is an in situ remedial technology that reduces concentrations of volatile constituents in petroleum products that are adsorbed to soils and dissolved in groundwater. This technology, which is also known as "in situ air stripping" and "in situ volatilization," involves the injection of contaminant-free air into the subsurface saturated zone, enabling a phase transfer of hydrocarbons from a dissolved state to a vapor phase. Advantages of in situ bioremediation: Minimal site disruption. Minimal exposure to public. Low costs. Disadvantages Time consuming process. Seasonal variation of microbial activity- prevailing environmental conditions. Problematic application of treatment additives( nutrients, surfactants and oxygen). The microorganisms act well only when the waste materials help to generate energy and nutrient to build up more cells. When the native microorganisms lack biodegradation capacity, genetically engineered microorganisms (GEMS) may be added to the site during in situ bioremediation. Stimulation of microorganisms is preferred over addition of GEMS. Ex situ Bioremediation (Contaminated soil is excavated and treated at another site). Ex situ bioremediation process may be implemented to treat contaminated soil or water that is removed from a contaminated site. Ex situ bioremediation technology includes most of disadvantages and limitations. It also suffers from costs associated with solid handling process e.g., excavation, screening and fractionation, mixing, homogenizing and final disposal. On the basis of phases of contaminated materials under treatment ex situ bioremediation is classified in to two Solid Phase system( including land treatment and soil piles) i.e., composting and land farming, biopiling. Slurry phase system(involving treatment of solid- liquid suspensions in bioreactors). Land farming: In this technique the Contaminated sludge, soils or sediments are spread on fields and cultivated in the same way as a farmer might plough and tilled, fertilize agricultural land. In land farming soil is removed and take to another area where it is spread out in thin layers, about 18 inches deep. Then biological materials (microbes) are added and the land is tilled to aerate. Care should be taken to prevent contamination of ground by leachate from the tilled zone. The technology is very simple and inexpensive, but it requires a large amount of space. Solid Phase system Composting: In this method the contaminated Soil is mixed with other organic Materials (straw, woos chips etc) and left in a pile for the Organisms to metabolize the Contaminant. Periodic mixing or turning is applied in order to ensure adequate aeration. Used for highly contaminated sites. Biopiles : In this method the contaminated soil is placed in a pile with alternating vent layers to provide the oxygen necessary for bacterial growth. Leachate is controlled by placing a linear beneath the pile and may be recirculated onto the pile. Biopile must be of sufficient size to prevent rapid heat and moisture loss. Advantages: Biopiles do not contaminate additional soil. Odour and dust are minimized and easily controlled Temperature and moisture levels are easily controlled Space required to built a biopile is many times smaller than that required to landfarm the same material. Slurry Phase System The contaminated solid materials (soil, degraded sediments etc.,), microorganisms and water formulated into slurry are brought with in a bioreactor i.e., fermentor. Thus, slurry phase system is a triphasic system involving three major components. They are water, suspended particulate matter and air (O2). Here water serves as suspending medium where nutrients, trace elements, pH adjustment chemicals and desorbed contaminants are dissolved. Bioreactor: A growth chamber or a vessel (fermentor) for cells or microorganisms. The cells or cell extracts carry out biological reaction in a bioreactor. Processes can be monitored, regulated and modeled mathematically very precisely. Biologically there are three types of slurry phase bioreactors. They are: Aerated lagoons Low shear air lift reactor (LSALR). Fluidized- bed reactor Aerated Lagoons or Slurry phase lagoon: Aerated lagoons are used for treatment of small common municipal waste water. Nutrients and aeration are supplied to the reactor. Mixers are fitted to mix different components and form slurry, where as surface aerators provide air required for microbial growth. Slurry-phase lagoon Low Shear Airlift reactors (LSARs): The LSARs are useful when waste contains volatile components. LSARs are cylindrical tanks which is made up of stainless steel. In this bioreactor pH, temperature, nutrient addition, mixing and oxygen can be controlled as desired. Shaft is equipped with impellers. It is driven by motor set up at the top. The rake arms are connected with blades which are used for resuspension of coarse materials that tend to settle on the bottom of the bioreactor. Air diffusers are placed radially along the rake arm. Baffles make the hydrodynamic behaviours of slurry phase bioreactors. Low shear air lift bioreactor Fluidized bed reactor: Fluidized bed reactors (FBR) Very small particles of 0.2 to 0.3 mm size like sand, carbon, fly ash, anthracite, glass, calcinated clay, etc., can function as the solid support medium for biological slime film development. These particles, being small, are maintained in suspension by the upward flow of liquid being treated. These support particles neither sink nor flowout. The reactors are generally cylindrical with perforated distribution plates. Entry sections are tapered or conical. The support particles are allowed to fluidize, but not to clump. Fluidized bed reactor Advantages:  Compact and small reactor which saves space.  Easy installation and transportation  Quick and economical expansion alternative in treatment plant.  Addition of modules to the existing system can be easily done.  Suitable for high strength industrial waste water.  Less cost for operation and also low capital.  Higher reactor biomass concentration.  High efficiency. References: Microbial Bioremediation by P. Rajendran & P. Gunasekaran Environmental Biotechnology theory and application by Gareth, John Wiley and sons. Websites: http://www.clu-in.org/products/costperf/BIOREM/FRENCH.HTM http://www.biostim.com/assests/images/bioremediation http://www.ecocycle.co.jp/e-bioremediation.html http://www.clw.csiro.au/research/urban/protection/remediation http://www.sciencedaily.com THANK YOU…..