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Bioremediation : 


What is Bioremediation? : 

What is Bioremediation? Bioremediation Biology “Remediate”= To solve a problem Bio-Remediate= to use biological organisms to solve an environmental problem Bioremediation   The collective range of clean up methods by using natural microorganism (such as bacteria, plant, Fungi, etc.) 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.

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Bioremediation • A technology that encourages growth and reproduction of indigenous microorganisms (bacteria and fungi) to enhance biodegradation of organic constituents in the saturated zone • Can effectively degrade organic constituents dissolved in groundwater and adsorbed onto the aquifer matrix • Generally requires a mechanism for stimulating and maintaining the activity of the microorganisms, e.g., addition of an electron acceptor (oxygen, nitrate); nutrients (nitrogen, phosphorus); and an energy source (carbon) Biodegradation • Biodegradation – microbially catalyzed reduction of complex of chemicals • Mineralization - conversion of an organic substrate to inorganic end products • Growth-linked metabolism – biodegradation provides carbon and energy to support growth • Maintenance metabolism - biodegradation not linked to multiplication, but to obtaining carbon for respiration to maintain cell viability; take place only when organic carbon concentrations very low

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Structure Volatility Chemical activity Affects fate of cpds

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Xenobiotic:organic compounds not found in the environment. Includes insecticides, pesticides, herbicides but not petrochemicals (products of living material) eg. DDT, HCH Biomagnification:increase of a toxic compound due to accumulation in tissues in organisms (if they are part of a food chain) as compared with levels found in the environment. Eg. DDT Mineralization:complete degradation to CO2 and water and other inorganic compounds and partial degradation refers to breakdown to an intermediate stage. Persistent organic cpds undergo degradation under certain circumstances Recalcitrant compounds are not degraded under any circumstances Bioconcentration factor: concentration of a pollutant from the environment and the factor is the concentration in an organism compared with that in the environment. Terms used in Bioremediation

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PRINCIPLES OF BIOREMEDIATION Bioremediation is based on the idea that organisms are capable to take in things from the environment and use it to enhance their growth and metabolism. With this unique characteristic lay the fundamental principle of Bioremediation, to use microorganism to take in contaminated substances from the environment or convert it to a nontoxic form. Bacteria, Protista, and fungi are well known for degrading complex molecules and transform the product into part of their metabolism.


PROCESS OF BIOREMEDIATION Microbes releases enzyme to break down the contaminant into digestible pieces The contaminant of organic substances is ingest and digest as food along with other energy source by the cell. GOAL Degrade organic substances that are hazardous to living organisms and degrade the organic contaminants into inert products. So only harmless biological wastes are all that remain of the contaminant.

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Xenobiotic Compounds Biocides, Herbicides, pesticides, weedicides Polychlorinated biphenyls (PCBs) are used in hydraulic fluids, plasticizers, adhesives, lubricants, flame retardants and dielectric fluidsin transformers released during production from spillage and disposal. Organochlorine or chlorinated compounds: trichloroethenes, pentachlorophenols, carbon tetra chloride Polyaromatic hydrocarbons (PAHs), contaminants from them like dioxins, dibenzofurans Accumulate in environment because: Degraded very slowly or are permanent Half life can be measured in years Persistence is influenced by structure and properties of the molecule (chemical and physical) Complexity of structure: presence of halogens (more the no. more persistent, less soluble in aqueous phase, increased toxicity), number of isomeric forms eg. PAHs Little information on toxicity and long term effects Synthetic Compounds

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Petrochemical Compounds Crude Oil is a complex and variable mixture of organic cpds Accumulated underground as a result of anaerobic degradation of MO for a long time under conditions of high temperature and pressure organic material is converted to natural gas, liquid crude oil, shale oil, tars. Only crude oil is liquid and if not contained it will escape to surface and volatiles will evaporate Major cpds in crude oil are hydrocarbons Methane Aliphatic n-alkanes, pentanes, hexanes, heptanes, cycloparaffins Monocyclic aromatics: benzene, toluene, ethylbenzene, xylene (BTEX) Polycyclic hydrocarbons (PAHs; 5-35%): napthalene, anathracene, phenanthrene Heterocyclic cpds containing S, N2 and heavy metals Tars and bitumen Straight, branched chains, condensed rings, aromatic rings Aromatic HC: 1. monocyclic HC (BTEX) 2. PAHs (napthalene, phenanthrene, anathracene) Diverse range of molecular structures

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Petrochemical Compounds Crude oil LMW HMW Light oils Heavy oils Refining breaks PAH to monocyclic HC like BTEX Product Carbon No. Boiling Point (oC) Methane 1 -160 Petroleum ether 4-6 20-60 Light naptha 6-10 60-100 Petroleum (gasoline) 3-8 40-205 Paraffin (kerosene) 10-14 165-200 Diesel 15-20 175-365 Fuel Oil 20+ 350+ Diesel requires higher temp to burn than petroleum

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Inorganic Waste Metals Inorganic cpds Mining Smelting Electroplating Farming Toxic at high concentrations Eg Cu, Hg As, Cn, F, Sb Minamata disease

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Bioremediation Strategies Old treatment involved “Immobilization, removal by dig and dump, thermal and solvent treatment” Bioremediation is cheaper than physical and chemical methods and can deal with lower concentrations (may take longer) Use of indigenous microbial populations Biostimulation:Encourage the indigenous population Bioaugumentation: addition of adapted or designed inoculants Addition of GMO Phytoremediation

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Use of indigenous microbial populations Naturally occuring hydrocarbon degrading MO (bateria, fungi, even cyanobacteria and algae). HC soil have more MO than uncontaminated soils Fate of a cpd is affected by 2 factors: Factors affecting growth and metabolism of MO Factors imposed by cpd: chemical structure, availability solubility and effects of photochemistry. Rate of degradation is dependent on chemicals structure of the cpd aliphatic and monocyclic aromatics simple to degrade but PAHs which are complex difficult. persistence increased if cpds or breakdown products are toxic persistence is enhanced by presence and number of halogens Bioavailibility: non availibility of cpds or intermediates for microbial degradation. Can be influenced by soil structure, porosity, composition and solubility. Eg. Certain intermediates can bind irreversibily to clay particles In sea miscibility and solubility (use of surfactants can render breakdown of some cpds and make them bioavailable.

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Biostimulation:Encourage the indigenous population If MO cannot use pollutant as their sole source of energy and carbon other growth substrates will be needed. In other cases MO may use a growth substrate in preference to the HC. Marine MO may need supplementation with N2 or P etc. Factors affecting growth of MO: presence of other biodegradable organic material Presence of N2, P containing inorg cpds O2 level Temp. pH Water and soil moisture No. and type of MO Presence of heavy metal or salt Aerobic is faster than anaerobic O2 presence enhances growth of MO Low temp decreases rate of degradation pH affects growth and solubility N2 and P in fertilizer enhances growth of MO Presence of Metals (conc. and type) inhibit growth of MO

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Biostimulation:Encourage the indigenous population Co-metabolism: addition of a second carbon source stimulates degradation of pollutant. Problem with this is that substrate added should be evenly distributed in the soil Sometimes breakdown products are difficult as they reversible or irreversibly bind to clay. Eg TNT Pollutants cnn be insoluble and hence non bioavalible. Addition of surfactants can breakdown such products and make them bioavailibleeb. Phenanthrene,pentachlorophenol have been shown to increase biodegradation using Trtiton X 100 and Triton- A45 Extraction with liquid CO2 successful for removal of diesel from soil Soil washing for sparingly soluble like PCP Crude oil mobility can be increased by addition of biodiesel

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Bioaugumentation: addition of adapted or designed inoculants Addition of selected MO to contaminated sites to supplement indigenous populations Generally introduced MO decline rapidly after growth due to faliure to compete with already existing MO. Bioaugumentation has been considered for crude oil with mixed results. Vast diffrence b/w lab and field. If want to see fate of introduced MO use techniques like DGGE, TGGE abd 16srRNA Causes of Bioaugumentation failing: Limited no of MO Conc. Of pollutant or contaminant not sufficient to support growth of MO Envt contain growth inhibiting substances Predation (eg protozoa) Added MO may use other substance other than pollutant MO not able to penetrate enough to reach contaminant

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Addition of GMO Camphor CAM Octanol OCT Xylene XYL Napthalene NAH Combined in one construct Combined in one construct Combined in one construct CAM OCT XYL NAH “Superbug” Pseudomonas putida

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Bioremediation on Land Land Farming: mixing soil by mechanical tiling. Ploughing causes O2 levels to increase and distributes contaminants more evenly to increase rate of biodegradation. Nutrients can also be added Used when contaminants are shallow. Area can be lined to prevent leaching into groundwater system. Bioventing: an in situ technique where the subsurface is aerated in order to promote biological activity of the microorganisms ...increased O2 supply with vapour extraction. Vacuum is applied at some depth in the contaminated soil and sweeps out any volatile organic cpd. Incerase in air supply increases natural degradation by aerobic MO. DISADV: only effective for volatile cpds and soil should be permeable. Vapour extracted may need treatment eg biofilters IN SITU

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Biosparging: to increase biological activity of soil supply of O2 is given by sparging air into the soil. DISADV: expensive process but on site reduces costs IN SITU

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EX SITU Composting: Solid phase bioremediation carried out after extraction Straw, bark, wood chips + mixed contaminating soil piled in heaps Rise in temp to 60oC Microbial Activity Thermophilic bacteria Used in diesel contaminated soils High temperature of composting Increases diesel availability for microbes Use sewage or compost 80% pollution removed by day 10 Organic waste like Vegetable peeling, garden waste (33-75%) Temperature of 60-70oC after 6-22 days with turning every 7 days 84-86% contamination removed by day 40

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EX SITU Biopile: Soil is heaped into piles within a lined area to prevent leaching Piles are covered with polythene and liquid nutrients applied to surface Aeration improved by suction to base of pile, leachate is collected by pipes at base and can be recycled Used when space is limited and vapour emissions need to be restricted (biofilter) Solid phase bioremediation Moisture, heat, nutrients, oxygen, and pH can be controlled to enhance biodegradation.

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Applicability: Biopile treatment has been applied to treatment of nonhalogenated VOCs and fuel hydrocarbons. Halogenated VOCs, SVOCs, and pesticides also can be treated, but the process effectiveness will vary and may be applicable only to some compounds within these contaminant groups Limitations Factors that may limit the applicability and effectiveness of the process include: Excavation of contaminated soils is required. Treatability testing should be conducted to determine the biodegradability of contaminants and appropriate oxygenation and nutrient loading rates. Solid phase processes have questionable effectiveness for halogenated compounds and may not be very effective in degrading transformation products of explosives. Similar batch sizes require more time to complete cleanup than slurry phase processes. Static treatment processes may result in less uniform treatment than processes that involve periodic mixing. Biopile:

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Bioreactor: Soil extracted from a contaminated soil or site can be treated as solid waste (Slurry 10-30%) or as liquid lechate in bioreactors of different designs A bioreactor gives control of parameters like temp., pH, mixing, O2 supply to improve degradation rates Solid bed Fluid bed Stirred tank Bioreactors can be used for sewage treatment,contaminated wastewater, soil slurries, metal removal and solid treatment Biorector type Sewage Metal removal Solid state Stirred tank ✔ ✔ ✔ Activated sludge ✔ Trickling filter ✔ Rotating biological contractor ✔ ✔ Fluid bed ✔ ✔ ✔ Fixed bed ✔ ✔ Upflow anaerobic sludge blanket (UASB) ✔ ✔ Biopile ✔ Rotating drum ✔

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Biomass is fixed to some form of solid medium Trickling filter, rotating drum contractor, upflow fixed film, fluid bed reactor Biomass is freely suspended Stirred tank, activated sludge, UASB Contaminated groundwater leachates Wastewater Contaminants at low conc Low loads on system Slow degradation rate Slow biomass growth An immobilized system like upflow fixed film or fluid bed bioreactor Activated sludge system is used to remove xenobiotics like 2,4-DCP by augumenting with a special mixed culture…also done for treatment of phenols, chloroanailine, chlorinated solvents, aromatic HC, petroleum HC.

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An immobilized system like upflow fixed film or fluid bed bioreactor Immobilization gives high surface area and operates continuously with high biomass content Microbial culture is immobilized on an inert system If contaminated material is solid can be treated in a stirred tank in soil slurry system OR Fixed bed, biopile or rotating drum design can be used Sulphate reducing bacteria are used to produce metal sulphides and anaerobic condition are neeed in USAB

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Novel technologies: Use of ANTIBODIES to remove a specific pollutant; COST IS TOO HIGH SO USE RESTRICTED NANOSCALE IRON PARTICLES: 1-100nm, 10X less than bacteria are injected into contaminated site. Particles are small enough to permeate b/w soil particles and degrade contaminants. They can be injected under pressure. PERMEABLE REACTIVE BARRIER: to treat acid mine drainage. It’s a trench filled with porous material like peat in an outflow from a mine. Porous material acts as an immobilization matrix for MO that can degrade or ppt contaminants. Stone chips and compost can be used as barrier: Stone chips can be used as a surface for MO to colonize and compost provides nutrients.

Phytoremediation : 

Phytoremediation Plants have been commonly used for the bioremediation. Process called Phytoremedation, which is to use plants to decontaminated soil and water by extracting heavy metals or contaminants . HOW DOES IT WORK Plants that are grown in polluted soil are specialized for the process of Phytoremedation. The plants roots can extract the contaminant, heavy metals, by one of the two ways, either break the contaminant down in the soil or to suck the contaminant up, and store it in the stem and leaves of the plant. Usually the plant will be harvest and removed from the site and burned. Phytoremediation Process is use under satisfy environmental regulation and costs less then other alternatives. This process is very affective in cleaning polluted soil.

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Phytoremediation Minimal disruption, no disruption to topsoil Effective with low levels of mixed contamination Inexpensive DISADV Process is slower than bioremediation methods Retardation of growth of plants due to contaminants Plants which accumulate contaminants is a hazard to wildlife and food chain Bioconversion factor is 1 for most plants BF needs to be 20 or above for phytoremediation to reduce contamination by 50% More the BF more the rate of phytoremediation Hyperaccumulators: plants that accumulate high concentration of metals 50-100 times more than normal plants but biomass produced maybe less 10,000mg/g for Zn and Mn 1000mg/kg for Co, Cu, Ni, As 100mg/kg for Cd Involves adsorption, transport and translocation to areas where large quantities of metal can be stored. [single gene for n accumulation]

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Phytoaccumulation: storage of contaminant in plant Uptake and degradation of organic cpds Phytostabilization: transformation of one species into less toxic (Cr6+ to Cr3+) Methyl t-butyl ether (MTBE), TCE

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Rhizodegradation Rhizofiltration: uptake of metals or degradation of organic cods by MO in rhizosphere. Artificial wetland systems (Phragmites sp.) are used. Such plants can grow in brackish waters, O2 transfer from leaves to roots and hence large aerobic MO in rhizosphere. Roots act like a microbial film reactor and sequesters metal and organic cpds. These have been used to clean mine leakage contaning heavy metals, municipal wastewater, dairy waste, N2 and P waste from wastewater. Rhizostimulation: stimulation of plant growth by rhizosphere by providing better growth conditions or reduction in toxic cpds. Free living bacteria are growth promoting which are promoted due to the nutrients exuded by plants in rhizospere. These help in uptake of contaminants and alsoprevent pathogens, stimulate plant growth. Eg. Mycorhizzal fungi Phycoremediation: use of micro- and macroalgae to remove acids, metals, organic pollutants, CO2 sequestration, biodegradation of xenobiotic cpds and as part of biosensors. Applications of genetic engineering for phytoremediation

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Metal Bioremediation Chemical and biological process cannot degrade metals hence any process must concentrate metals so that it can be contained or recycled Cd, Zn, Cu, Pb, Hg Biosorption Biological material can adsorb variety of metals. There are several responses of the microbial cell towards high concentration of metals Exclusion of metal from cell Energy dependent efflux of metal from cell Intracellular sequestration by specific proteins like metallothioneins Extracellular sequestration on cell wall or extracellular polysaccharides Chemical modification of metal

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Biosorption Metal uptake from wastewaters can be active or passive or both. Passive is independent of cellular metabolism, is rapid and is completed within 5-10 min, unaffected by metabolic inhibitors, reversible and can occur in living or dead material. Active slow, dependent on cellular metabolism, affected by inhibitors, uncouplers, temp. Metals can be complexed with specific proteins (metallothioneins) or contained in a vacuole. Used for removal of metals from waste streams, mine waters. Biomass used must be inexpensive eg sewage slugde, marine alga, seaweed, living or dead material Metal binding proteins are highly expressed Several bioreactors like fixed bed, fluid bed and rotating disc reactors are used





CASE: Crude oil spill, Bemidji, Minnesota : 

CASE: Crude oil spill, Bemidji, Minnesota In the year 1979, in Bemidji, Minnesota a pipeline carrying crude oil suddenly exploded and releasing an enormous amount of oil. As a result of the oil spill, toxic chemicals were released which rapidly degraded the microbial population. The plume of contaminated ground water stopped enlarging after a few years as rates of the microbial degradation came in to balance with rates of the contaminant leaching.



BIOREMEDIATION: A Choice to Make : 

BIOREMEDIATION: A Choice to Make ADVANTAGES WHY USE BIOREMEDIATION 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 Transform pollutants instead of simply moving them from one media to another Perform the degradation in an acceptable time frame DISADVANTAGES POTENTIAL PROBLEMS Cost overrun Failure to meet targets Poor management Climate Issue Regulatory compliance concern Release of contaminants to environment Unable to estimate the length of time it’s going to take, it may vary from site. 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 many be more toxic then it’s parental form