Chapter 4A - Biodegradation of Specialty Compounds PDF

CHAPTER 4A: MICROBIAL DETOXIFICATION OF SPECIALTY CHEMICALS  BIOREMEDIATION can effectively degrade the following contaminants: Hydrocarbons with carbon chains ranging from C-5 to C-40 Benzene, xylene and toluene TCE (Tri-chloro-ethylene) PAH (Poly-aromatic- hydrocarbons) PCB (Poly-...

CHAPTER 4A: MICROBIAL DETOXIFICATION OF SPECIALTY CHEMICALS  BIOREMEDIATION can effectively degrade the following contaminants: Hydrocarbons with carbon chains ranging from C-5 to C-40 Benzene, xylene and toluene TCE (Tri-chloro-ethylene) PAH (Poly-aromatic- hydrocarbons) PCB (Poly-chlorinated- biphenyls) and other chlorinated compounds Fuel oils Fossil fuels - gasoline, diesel, aviation gas Condensate - leakage from pipelines Factors affecting the biodegradation of petroleum hydrocarbons a) Chemical composition and hydrocarbon concentration Classes of petroleum hydrocarbons: a) Asphaltenes (phenols, fatty acids, ketones, esters and porphyrins) b) Resins (pyridines, quinolines, carbazoles, sulfoxides and amides) c) Saturates Susceptibility of hydrocarbons to microbial degradation is in the following order: N-alkanes>branched alkanes>low m.w aromatics>cyclic alkenes  Alkanes are usually the easiest hydrocarbons to be degraded by conversion to alcohol via mixed oxygenase activity. The simpler aliphatics and monocyclic aromatics are readily degradable More complex compounds eg PAHs are not easily degraded and may persist for some time. The persistance is increased if the compound is toxic or its breakdown products are toxic to the soil microflora eg phenol and hydroquinone are the major products of benzene oxidation with the ability of hydroquinone to exert toxic effect as accumulated concentrations inhibit the degradation of other pollutants.  High m.w. aromatics, resins, and asphaltenes have a slow rate of biodegradation. The more complex and less soluble oil components are degraded more slowly than lighter oils. High concentrations of hydrocarbons in water means heavy undispersed oil slicks causing a limited supply of nutrients and oxygen resulting in the inhibition of biodegradation. Protection of oil from dispersion by wind and wave action in beaches, small lakes and ponds explains the presence of high concentrations of hydrocarbons in these places and the accompanied negative effects on biodegradation. Oil sludge contaminating the soil at high concentrations also inhibits Mos in their action. The quantity of crude oil spilled in soil influences the rate and total extent of disappearance of the soil in the environment. Physical state The physical state of petroleum hydrocarbons has a marked effect on their biodegradation. Crude oil in aquatic systems usually does not mix with seawater , floats on the surface , allowing the volatilization of the 12 carbons or less components. The rate of dispersion of the floating oil depends on action of the waves which in turn is dependent on the weather. Crude oil with a high proportion of light oils flow easily and dispersed easily in a short time. Due to Wind and wave actions, oil-in-water or water-in-oil (mousse) emulsions form which in turn increase the surface area of the oil and its availability for microbial attack. A low surface-to-volume ratio as a result of formation of large masses (plates) of mousse or large aggregates of weathered and undegraded oil (tarballs) inhibits biodegradation because these plates and tarballs restrict the access of Mos. One of the factors that limit biodegradation of oil pollutants in the environment is their limited availability to Mos. Availability of the compound for degradation within the soil plays a crucial factor in the determination of the rate of hydrocarbon degradation.  To solve the problem, surfactants are added to contaminated soils and sea water to improve access to the hydrocarbons, with different chemical dispersant formulations have been studies as means of increasing surface area and hence increasing breakdown of hydrocarbon pollutants. The concentration of dispersant and the dispersant/oil application ratio determine its effectiveness in enhancing the biodegradation of oil slicks. Not all dispersants enhance biodegradation. Factors affecting availability Soil structure Soil porosity Soil composition Solubility of the compound Soil particle size distribution also affects microbial growth, so that a soil with an open structure encourage aeration and thus the rate of degradation will be affected. Infiltration of oil into the soil prevent evaporative losses of volatile hydrocarbons which are toxic to Mos. Particulate matter reduce the effective toxicity of the components Physical factors a) Temperature b) Pressure c) Moisture Temperature Influence the petroleum biodegradation by i. its effect on the composition of the microbial community and its rate of hydrocarbon metabolism ii. On the physical nature and chemical composition of the oil. The decrease in evaporation of toxic components at lower temperatures can influence nonbiological losses mainly by evaporation. In some cases the decrease in evaporation of toxic components at lower temperatures was associated with inhibited degradation. The optimum temperature for biodegradation of mineral oil hydrocarbons under temperature climates is at 20-30C. most mesophilic bacteria perform best at 35 C , temperatures within 30- 40C maximally increase the rates of hydrocarbon metabolism. At low temperatures, the rate of biodegradation of oil is reduced due to decreased rate of enzymatic activities. Cold climates select for lower temperature indigenous Mos with Pressure In the deep-ocean environment, biodegradation occurs at very slow rate. Eg after 40-week high pressure incubation, 94% of the hexadecane was degraded by a mixed culture of deep-sea sediment bacteria under 1 atm and 495 or 500 atm at 4°C, the same amount that occurred after 8 weeks at 1 atm. Moisture Bacteria rely upon the surrounding water film when they exchange materials with the surrounding medium through the cell membrane. At soil saturation, all pores are filled with water. Soil moisture levels in the range 20-80% of saturation allow suitable biodegradation to take place. 100% saturation inhibits aerobic biodegradation due to lack of oxygen. Chemical factors Oxygen In most petroleum-contaminated soils, sediments and water, oxygen is the limiting requirement for hydrocarbon biodegradation the bioremediation methods for reclamation of these contaminated sites is mainly based on aerobic processes. bacteria and fungi use oxygenase enzymes for the breakdown of aliphatic, cyclic and aromatic hydrocarbons. Requiring oxygen. The availability of oxygen in soils, sediments and aquifers is often limiting and dependent on the type of soil and whether the soil is waterlogged. Oxygen concentration is the rate-limiting variable in the biodegradation of petroleum hydrocarbons in groundwater. Anaerobic hydrocarbon degradation occur at very slow rates and its ecological significance appears to be minor. Several studies have shown that anaerobic hydrocarbon metabolism can be important process in certain conditions eg. Biodegradation of some aromatic hydrocarbons such as BTEX compounds occur under a variety of anaerobic conditions. Anoxic biodegradation in the BTEX family of compounds, except benzene, can be mineralized or transformed cometabolically under denitrifying conditions. pH In soils and poorly buffered treatment situations, organic acids and mineral acids from various metabolic processes can reduce the pH. The overall biodegradation rate of hydrocarbons is generally higher under slightly alkaline conditions. Therefore need to maintain pH range of 7.0-7.5. The pH of soil is an important factor for anthracene and pyrene degradation activity of introduced bacteria Sphingomonas paucimobilis. A shift of pH from 5.2 to 7.0 enhanced degradation by the bacteria. Salinity Studies showed the rates of hydrocarbon metabolism decreased with increasing salinity due to general reduction in microbial metabolic rates. Biodegradation of crude oil was greatest at lower salinities and decreased at salinities more than twice that of normal saltwater. Water activity (Aw) Microbial degradation of hydrocarbons in the environment showed terrestrial ecosystems may be limited by the water available (A w= 0.0 – 0.99) for microbial growth and metabolism. Optimal rates for biodegradation of soil sludge in soil is at 30/90% water saturation. Aw in aquatic environment is stable at 0.98 and may limit hydrocarbon biodegradation of tarballs deposited on beaches. Nutrients Spilled oils contain low concentrations of inorganic nutrients. Thus limit the C/N or C/P ratios are high and often limit microbial growth. Addition of phosphorus and nitrogen in the oleophilic fertilizers can adjust the ratio of C/N or C/P and thus enhancing biodegradation of the spilled oil. The release of these products containing substantial amounts of N, P and other limiting compounds is slow. Thus the nutrient retention time is increased in contrast to water-soluble fertilizers which have a restricted retention time. Oleophilic fertilizers are essential in environments with high water exchange or if water transport is limited, and proved to be more effective than water-soluble fertilizers when the spilled oil resided in the intertidal zone. The effect of different nutrient combinations (C/N/P) on biodegradation of oil deposited on shorelines by monitoring the total number of bacteria, the metabolically active bacteria and oil degradation. The treatment result in increased degradation of oil compared to non-treated crude oil or crude oil. Rate of biodegradation of crude oil or gasoline in soil and Biological factors The rate of petroleum hydrocarbon biodegradation in the environment is determined by : a) The populations of indigenous hydrocarbon degrading Mos b) The physiological capabilities of those populations c) Other various abiotic factors that may influence the growth of the hydrocarbon-degraders. Hydrocarbon biodegradation depends on composition of microbial community and its adaptive response to the presence of hydrocarbons. Bacteria and fungi are the principal agents in hydrocarbon biodegradation. Bacteria are dominant in the marine ecosystems Fungi are dominant in freshwater and terrestrial environments. Hydrocarbon-utilizing bacteria and fungi are readily isolated from the soil and the introduction of oil or oily wastes into soil caused increases in in the numbers of both groups. Algae and protozoa has no ecologically significant role played by these groups in the biodegradation of hydrocarbons. What informations do we expect to get from Genera of bacteria mostly recovered form the Mos in the polluted site? contaminated soils : a) Microbial enumeration Pseudomonas not a direct measure of their activity in soils Enterobacter It provides an indication of microbial vitality Acinetobacter and/or biodegradative potential. Bacillus Streptomyces b) Biodiversity Rhodococcus In a crude petroleum oil contaminated soil, biodiversity indicates how well the soil In gasoline and diesel station soils, supports microbial growth. bacteria are the most dominant flora and the genus was Corynebactrium was the c) The distribution of the type and the predominant genus. number of Mos at a given site may help to Ways of obtaining more organisms characterize that site with respect to the adapted to hydrocarbon pollutants: concentration and duration of the a) The prevalence and widespread of contaminant. genus Pseudomonas in hydrocarbon- d) Fresh spills and/or high levels of polluted soils and reflect their potential contaminants often kill or inhibit large use against these hydrocarbon sectors of the soil microbiota , whereas contaminants soils with lower levels or old contamination b) Genetic manipulation. This allows the show greater numbers and diversity of transfer of degradative ability between Mos. Long duration contamination sites bacteria and particularly in soil. Thus a showed greater number s of Mos, whereas rapid adaptation of the bacterial fresh spills reduced the bacterial number in population to a particular compound is the crude oil polluted soil. promoted and the pool of hydrocarbon- catabolizing genes carrier organisms within the community is clearly enhanced. Therefore the number of hydrocarbon utilizing organisms would Biodegradation of selected compounds i. Alkanes a) Hydrocarbons The n-alkanes are the most Billions of tons of petroleum are produced biodegradable of the petroleum per year worldwide. And large amounts of hydrocarbons. petroleum products end up polluting both Normal alkanes in the C5 to C10 marine and terrestrial environments. range are inhibitory to many Where do this petroleum discharge come hydrocarbon degraders at high from? concentrations because as solvents 90% of the total petroleum hydrocarbon they disrupt lipid membranes. discharges come from low-level routine Alkanes (C20-C40 range) are discharges (urban runoff, effluents, oil hydrophobic solids; their solubility treatment of roads, etc). interferes with their biodegradation. 10% come from accidents eg tank In alkane degradation, the disasters, pipleine breaks, and blowouts. monooxygenase enzyme attacks the Petroleum hydrocarbons are intermediate terminal methyl group to form an between highly biodegradable and highly alcohol. The alcohol is further recalcitrant compounds. oxidized to an aldehyde then to a Hydrocarbons are classified as; fatty acid. i. Alkanes (normal and iso) The fatty acid is further degraded by ii. Cycloalkanes beta oxidation of the aliphatic chain. iii. Aromatics The degradation of alkanes produces iv. Polycyclic aromatics oxidized products that are less v. Asphaltines volatile than the parent compounds. vi. Resins The parent alkanes are highly volatile There is a variety of hydrocarbons based and may be removed from soil on chain length, chain branching, ring primarily through stripping under condensations, interclass combinations, aerobic conditions. and presence of oxygen, nitrogen and sulfur-containing compounds. The biodegradability of these Alkenes Aromatics location of the unsaturated linkage is a Aromatic compounds have structures based factor that might influence the on the benzene molecule. degradation of the alkene. 1-,2-,3-,4- and 5-ring compounds and alkyl- Eg 1-alkenes, where the unsaturated substituted aromatics are present in bond is on the first carbon., are more petroleum. degradable than alkenes with an internal Benzene, toluene, ethyl-benzene, and the double bond. three xylenes (collectively known as BTEXs) Either the double bond is oxidized, giving are water soluble and the most mobile rise to a diol, or the saturated chain end is components of conventional gasoline. oxidized. Most potentially hazardous, especially benzene, being a carcinogen. Cycloalkanes BTEXs are often used as indicators of soil Also known as alicyclic hydrocarbons. and groundwater contamination, especially Are less degradable than their straight – from leaking underground storage tanks. chain alkanes but more degradable than Biodegradation of an aromatic molecule the polycyclic aromatics (PAHs). Part of the involves 2 steps : 1. activation of the ring 2. decrease in biodegradability is due to ring cleavage decreased solubility. Activation involves the incorporation of Alkyl-substituted cycloalkanes are more molecular oxygen into the ring. i.e. readily degraded than nonsubstituted dihydroxylation of of the aromatic nucleus hydrocarbons, and cycloalkanes with long using the enzymes oxygenases. –chain side groups are more easily Dihydrodiols are further oxidized to degraded than those with methyl or ethyl dihydroxylated derivatives eg catechols groups. which are precursors to ring cleavage. Are usually degraded by oxidase attack to Catechols are oxidized, then the compounds produce a cyclic alcohol which is are then degraded to form acids which are dehydrogenated to a ketone. readily utilized by Mos for cell synthesis and Cycloketones and cycloalkane-carboxylic energy. acids are therefore the primary products of Polycyclic aromatic hydrocarbons (PAHs) Asphaltines and resins Are produced during high-temperature Are high molecular weight compounds industrial operations eg. Petroleum refining, containing nitrogen, sulfur and oxygen. coke production and wood preservation. Have complex structural arrangements They are common contaminants in industrial composed of hydrocarbon chains and and uncontrolled hazardous –waste sites. nitrogen, sulfur and oxygen atoms linking An increase in molecular weight and polycyclic aromatic stacks which include number of ring structures yields decreased nickel and vanadium. solubility and volatility and increased Compounds in these groupings are adsorption capacity. recalcitrant to biodegradation due to their Are degraded, one ring at a time, by similar insolubility and the presence of functional mechanisms as the ones used for aromatic groups that are shielded from microbial attack compounds. by extensive aromatic ring structures. Biodegradability of PAHs tends to decrease with increased numbers of rings and with increasing numbers of alkyl substituents. The enzymes required for the prokaryotic degradation of PAHs can be induced by the presence of lower molecular weight aromatics eg naphthalene. The high molecular weight might be resistant to microbial degradation when low molecular weight PAHs are not present. Fungal degradation of PAHs is environmentally significant since some of the products have been implicated as toxic forms in higher organisms. Biodegradation of certain PAHs results in low molecular weight compounds having an increase in volatility.  The biodegradation rate is also dependent on Biodegradation of halogenated aliphatic compounds the type of the halogen in the compound. Halogenated aliphatic compounds are Halogens in their decreasing common contaminants of groundwater and electronegativities : F, Cl, Br, I. Microbially mediated reactions of chlorinated hazardous-waste sites. Industrially important halogenated aliphatic compounds include substitutions, aliphatics include chlorinated and oxidations, and reductions. Dehalogenation of the molecule is usually brominated alkanes and alkenes in the C1 to C3 range. the first step with compounds containing a Halogenated compounds are generally short alkyl chain. When the alkyl chain is long, the halogen no more resistant to microbial attack and tend to persist in the environment. longer influences the oxidation of the terminal The halogen atoms on the molecules carbon atom. In this case oxidation of the increase the oxidation state of the carbon terminal methyl group is the first step atom, and aerobic processes are resulting in a halogenated sliphatic alcohol. In substitution reactions, the halogen is energetically less favourable for the highly halogenated compounds. substituted by a hydroxyl group : Anaerobic degradation is more favourable. R---X + H2O R OH + HX Physicochemical processes eg. Stripping and adsorption are often more reliable and Eg is the dehalogenation of dichloromethane : effective than bioremediation for CH2Cl2 + H2O [HOCH2Cl] + HCl halogenated compounds owing to their slow degradation rates.many halogenated compounds are rather easily degraded. Eg dichloromethane, chlorophenol, and ortho-, HCHO + 2H+ + 2Cl- meta-, and para-chlorobenzoate. Organic compounds generally act as The third type of reaction, reductive electron donors; but due to the dehalogenation, occurs in anaerobic electronegativity of the halogen environments. Either a halogen is substituted substituents, polyhalogenated compounds by a hydrogen atom or 2 halogen atomsare removed, forming a double bond (dihalo- R X + H+ + 2e RH + X- C C C C + 2X- + 2 e- X X Dihalo-elimination can occur in either anaerobic and aerobic environments. BIODEGRADATION OF HALOGENATED Eg monochlorinated benzoates are all AROMATIC COMPOUNDS biodegraded to CO2 aerobically. Are common contaminants of soil, Reductive dechlorination of groundwater and hazardous-waste sites. pentachlorophenol (PCP) is shown below : Industrially important halogenated The products are 3, 4, 5-trichlorophenol, 3,5- aromatics include solvents, lubricants, dichlorophenol and 3-chlorophenol. pesticides (DDT, 2,4-D, 2,4,5-T), Substitution of the halogen by a hydroxyl plasticizers, polychlorinated biphenyls group occurs in para-substituted (PCBs), which were widely used as monohalogenated benzoates and PCP. insulators in electrical transformers and The product of PCP degradation is capacitors, and pentachlorophenol, a tetrachloro-p-hydroquinone, which can be wood preservative. degraded only under anaerobic conditions. Both the number and position of halogens are important in determining the biodegradability of halogenated aromatic compounds. The more halogen substituents the compound has, the more likely it is to undergo reductive dehalogenation in reducing environments. Biodegradation may occur by dehalogenation of the ring structure by oxidation, reduction or substitution. Ring cleavage can precede dehalogenation, generating halogenated aliphatic compounds. Reductive dehalogenation occur under methanogenic conditions for chlorinated benzoates, PCBs, PCP, the pesticide 2, 4, 5-T, chlorophenols, and 1, 2, 4- trichlorobenzene. Insecticides, herbicides, Fungicides SOIL RECLAMATION - OIL Petroleum hydrocarbons SPILL CLEANUP - Polychlorinated biphenyls and HYDROCARBON halogenated aromatics BIOREMEDIATION Industrial solvents and other wastes Bioremediation is an economical Xenobiotics and safe method for cleaning up oil spills and bioremediating soils contaminated with petroleum hydrocarbons and dangerous organic compounds. The bioremediation process utilizes beneficial microbes, surfactants, micronutrients and bio-stimulants to decompose contaminants transforming them into harmless byproducts, i.e. water and carbon dioxide. Example Pollutants Polychlorinated biphenols Pollutants that are being studied for PCBs are used in industrial bioremediation potential are listed applications, are very recalcitrant, below. and many are known carcinogens. Petroleum byproducts Chlorinated solvents BTEX - benzene, toluene, Chlorinated solvents are used ethylbenzene, and xylene - are extensively as cleaning agents. byproducts of petroleum products. Plumes have been found to The biodegradability of these contaminate groundwater below compounds is relatively well known dry cleaners in many places, and remediation can be achieved by including Davis, Ca. Many creating favourable conditions for chlorinated solvents are BTEX degrader's growth carcinogenic. Trichloroethylene Methyl tert-butyl ether (TCE) can be degraded to vinyl MTBE is a gasoline additive chloride under anaerobic introduced to replace lead. MTBE conditions. Vinyl chloride, in turn, raises the oxygen content of fuel, needs different conditions to allowing for more complete transform, and this should be combustion and less emissions. seriously considered due to its MTBE, however, is highly soluble, high toxicity does not adsorb well in soil and can therefore move quickly through soil and into groundwater Polycyclic aromatic compounds Dechloromonas aromatica PAHs are found in high concentrations at industrial sites especially sites that A soil bacteria genus which use or process petroleum products. are capable of degrading They are considered carcinogens and perchlorate and aromatic mutagens, and are very recalcitrant, compounds. pervading for many years in the Nitrosomonas europaea, natural environment. Nitrobacter hamburgensis Other contaminants include residuals , and Paracoccus from flares (perchlorate) and denitrificans explosives (TNT, RDX); metals Industrial bioremediation is (chromium, lead); plutonium and used to clean wastewater. uranium; potassium and nitrogen. Most treatment systems rely Much of the high levels of these on microbial activity to contaminants found in nature is a remove unwanted mineral result of human activity nitrogen compounds (i.e. ammonia, nitrite, nitrate). Example Microorganisms The removal of nitrogen is a Pseudomonas putida two stage process that Pseudomonas putida is a gram- involves nitrification and negative soil bacterium that is denitrification. involved in the bioremediation of During nitrification, toulene, a component of paint ammonium is oxidized to In anaerobic conditions, nitrate Deinococcus radiodurans produced during ammonium Deinococcus radiodurans is a oxidation is used as a terminal radiation-resistant extremophile electron acceptor by microbes like bacterium that is genetically Paracoccus denitrificans. The result engineered for the is dinitrogen gas. Through this bioremediation of solvents and process, ammonium and nitrate, two heavy metals. An engineered pollutants responsible for stain of Deinococcus eutrophication in natural waters, are radiodurans has been shown to remediated. degrade ionic mercury and Phanerochaete chrysosporium toluene in radioactive mixed The lignin-degrading white rot waste environments. fungus, Phanerochaete Methylibium petroleiphilum chrysosporium, exhibits strong Methylibium petroleiphilum potential for bioremediation of: (formally known as PM1 strain) pesticides, polyaromatic is a bacterium is capable of hydrocarbons, PCBs, dioxins, dyes, methyl tert-butyl ether (MTBE) TNT and other nitro explosives, bioremediation. PM1 degrades cyanides, azide, carbon MTBE by using the contaminant tetrachloride, and as the sole carbon and energy pentachlorophenol. White rot fungi source degrade lignin with nonselective extracellular peroxidases, which can Metabolic Pathways Microorganisms use a wide range of metabolic pathways to harvest energy from their environment. In some cases, pollutants serve as the carbon and energy source for microbial growth, while in other cases, pollutants serve as the terminal electron acceptor (ex. perchlorate degradation). This manifests itself in the diverse ability of microbes to transform and degrade toxic molecules. Polychlorinated Biphenyls (PCBs) Metabolism of polychlorinated biphenyls is generally proceed through the addition of two oxygens to the aromatic ring, followed by ring cleavage as seen in the metabolic pathways diagram. Energy is obtained through the oxidation of the large hydrocarbons. Phanerochaete chrysosporium, the white rot fungus described earlier, is thought to have the ability to degrade PCB by non-selective means.  Polycyclic aromatic compounds (PAHs) PAHs in contaminated soils can be treated Examples of PAHs are seen below: with bioremediation. The oxidation of PAH involves oxygenases (monooxygenases and dioxygenases). Fungi complete the process by adding an oxygen to the substrate PAH to form arene oxides and then enzymatically adding water to form trans-dihydrodiols and phenols. Bacteria mainly use dioxygenases, adding two oxygens to the substrate and then further oxidizing it to dihydrodiols and dihydroxy products. Ring oxidation is the rate limiting step in the reaction, and subsequent reactions occur fairly quickly, yielding the typical metabolic intermediate Catechol found in Lignin degradation as well as Gentisic and Protocatechuic Acids. Monitoring To monitor the bioremedation potential of a soil one can probe for the existence of specific degradation pathways in the soil community or monitor for specific enzymes involved in the process. There are two common ways to test for functional genes involved in the degradation of a compound. 1) Specific DNA hybridization probes can be used to indicate potential for the organisms to degrade the desired compound. 2) Specific RNA hybridization probes are used to indicate the expression of the functional genes in the environment. The actual change in pollutant concentration or degradation byproducts can also be monitored to determine the amount of pollutant removal. To determine if the degradation of a desired compound is the result of abiotic or biotic activity, controlled laboratory experiments are used. The concentration of a pollutant in a non-sterile microcosm containing soil from the environment of interest is compared to a sterile control. The sterile control shows the non-biological contribution to the disappearance of the pollutant due to, for example, adsorption to clay particles or volatilization. The non-sterile microcosm simulates the microbial contribution to the degradation of the pollutant in the natural environment, but also includes other abiotic mechanisms. The microbial contribution to pollutant disappearance is the difference between removal in the biologically active Bioremediation Applications Bioremediation was employed to treat the 1989 Exxon Valdez oil spill in Prince William Sound, Alaska. Hydrocarbon degrading microbes exist in marine systems because natural sources of hydrocarbon exists as a result of geological seeps and other sources. During the Exxon cleanup effort, the activity of these organisms was enhanced through the addition of nitrogen and phosphorus to oil laden beaches. This is an example of bio-stimulation. Current Research Pseudomonas putida has been found to be Methylibium petroleiphilum useful in the detection of certain chemicals, A motile, gram-negative facultative such as land mines. anaerobic bacterium, Methylibium On the grand scale, a linkage between the petroleiphilum]has been isolated because bacteria's ability to degrade TNT and the its ability to completely mineralize methyl explosive compound found in land mines has tert-butyl ether (MTBE), a gasoline inspired research to utilize P. putida as a way additive. of detecting land mines from soil content. Methylibium petroleiphilum is capable of consuming a diverse range of gasoline Nitrosomonas europaea derivatives as its sole carbon source, One possible treatment for the purification of including: methanol, ethanol, toluene, water has been the use of Trihalomethanes or benzene, ethylbenzene, and THM's. dihydroxybenzenes. Recent studies have linked these four Optimal growth of M. petroleiphilum chemicals, tricholormethane or chloroform, occurs at the soil subsurface with pH of bromomethane, dibromomethane and 6.5 and 30°C. The upper temperature limit dichlorobromomethane to colon cancer. of this bacterium is 37°C Because of its nitrogen oxidizing properties, Nitrosomonas europea has been studied under ammonia rich conditions and THM rich conditions, recognized as limiting reactants in the conversion of ammonia.  Industrial solvents and other wastes Sources of MSW: Wastes are classified as a) Residential a) Municipal b) Commercial b) Industrial c) Institutional c) Hazardous d) Construction and demolition sites d) Radioactive e) Municipal services (wastewater e) Mining treament , street cleaning, garden and f) Agricultural park landscaping). Wastes should be treated, stored and dispose. Wastes can be classified as : a) Organic b) Inorganic c) Microbiological Organic wastes: food, paper and cardboard, plastics, clothing, yard waste, and bone. Many of these organic wastes can be degraded. Inorganic wastes : silicates, sulphates, cyanides, iron, trace metals eg aluminum, cadmium, copper, lead, nickel and zinc, arsenic and its compounds. Metals do not degrade or decompose. When they are buried in a landfills, they can remobilize and threaten the environment.  Sources of organic wastes Industrial and Agricultural Municipal and commercial sources sources domestic sources Breweries Pigs Raw sewage Dairies Chickens Organic fraction of Food processing, Cattle municipal sludge packaging and Farmyards Food, paper shipping Cattle Activated sludge Chemical industries Crop residues Household waste Pharmaceutical Rotten products Grass Wineries Leaves Hazardous wastes Market wastes Oils Paper manufacture (pulps) Slaughterhouse  Food waste is highly biodegradable. Processes for waste treatment and Separation of these wastes from MSW and disposal industrial waste at the source can facilitate a) Incineration biodegradation processes and eliminate b) Disposal in a traditional landfill expensive and difficult sorting. c) Thermal desorption Hazardous wastes are separated from d) Microwave treatment industrial and municipal wastes. e) Solidification/stabilization Waste is considered hazardous if it is f) Vitrification ignitable, corrosive, reactive or toxic. Industrial wastes include organic sludges, oils and greases, solvents, heavy metal solutions, pesticides and herbicide wastes, PCBs and contaminated soils. Egs of household hazardous wastes : varnishes, paints, turpentines, motor oil, herbicides, pesticides, batteries and fertilizers. Radioactive wastes generated by specific industries eg nuclear reactors, research labs and medical facilities. In most countries, disposal of high-level waste is by burial in deep, stable geologic formations. In low level wastes are disposed of in a variety of ways but mainly by burial in caverns or clay formations. They become less radioactive over time due to radioactive decay. Insecticides Pesticide categories These specialty chemicals that are designed Halogenated aliphatic pesticides for their specific uses. Chlorinated cyclicaliphatic pesticides They are the pesticides and their Halogenated aromatic pesticides subcategories of insecticides, herbicides and Chlorinated phenylalkanoates fungicides, polychlorinated biphenyls and Halogenated aniline-based pesticides azo dyes. Carbamate pesticides Pyrethroid a) Pesticides Organophosphonate Have increased agricultural yields while impacting detrimentally on the food Hydrolytic reactions are the most significant chain of wildlife and humans. abiotic transformations. Toxic to some form of life. Hydrolytic reactions can be base-or acid- Some are not suitable for bioremediation catalyzed and often occur through but many can be biologically degraded. interactions with reactive chemical groups on mineral surfaces, reactive organic Chemical nature compounds and inorganic metals (eg Cu2+ ). Most pesticides are organic This rxn is necessary for microbial Can be classified according to common degradation. structural groups. Mos can catalyze the hydrolysis rxns through cometabolism. Degradation potential Degradation results in : The degradation principles are similar to a) Accumulation of metabolites the organic compounds. b) Incorporation of the pesticide compound Pesticides are subjected to both abiotic into the soil organic fraction and biotic transformation processes. c) Mineralization Many of the abiotic transformations result in partial degradation to products that can be further degraded by Mos.  In the bioremediation of pesticide- Development of degrading Mos for these contaminated soil, there are a few hard-to-degrade compounds require longer variables affecting degradation rate: time, months or years. i. availability of the pesticide to soil Bioremediation of these compounds is aided Mos. by developing specific seed cultures and This availability is a function of the inoculation of the soil or the bioreactor. adsorption affinity of the organic compound iv. Bioaugmentation of the soil to the soil. Bioaugment the soil increases the rate of The available pesticide for biodegradation degradation. is equivalent to the amount of pesticide Eg parathion degradation is enhanced by dissolved in the aqueous phase. the inoculation of an adapted two-member When ratio of water to soil solids culture. increases, total amount of available pesticide for degradation increases. v. Aerobic vs anaerobic condition for ii. Total biomass or number of Mos. degradation When the no. of Mos increases by Degradation is more rapid under anaerobic addition of nutrient and other than under aerobic conditions for many environmental controls, rate of pesticides, eg lindane, heptachlor, DDT, pesticide degradation also increases. aldrin and endrin. iii. Pesticide concentration This could be due to reductive For soil concentrations below 5 µg/L, dehalogenation by the anaerobes. the rate is first-order. At higher concentrations a biphasic breakdown occurs. For many sites contaminated with specialty compounds eg pesticides, PCBs and dyes,soil may not contain significant populations of degrading organisms. The degradation of certain compounds  There are only several kinds of reactions Incorporation of natural organic acid that initial their degradation. precursors and aromatic structures forming addition of a hydroxyl gp natural humus which is an irreversible Oxidation of an amino gp. complex. Oxn. Of a sulfur molecule These base compounds are biological Addn. of an oxygen to a double bond degradation by-products of natural organic Addn. of a methyl gp. matter. This is known as humification. Removal of a methyl gp. Humic compounds account for the bulk of Removal of a chlorine organic chemicals in soil and water. They are Chlorine migration extremely complex and of a varied structure. Reduction of a nitro gp. The phenolic structure is a major component of Replacement of a sulfur with an oxygen humic material and hazardous chemicals Cleavage of an ether linkage having this structure are potential candidates Metabolism of side chains for binding with natural soil material. Hydrolysis Halogenated aliphatic pesticides Refer handout on typical egs of the Are broad range fumigants used in agriculture. microbial initiated degradation of Eg 1,2-dibromo-3-chloropropane (DBCP) ,1,2- pesticides. dichloropropane, 1,3-dichloropropene and 2,3- dibromobutane. Complex formation Aerobic metabolism used to degrade methyl Not all microbial reactions result in bromide and many chlorinated aliphatic mineralization or even transformation to compounds. simpler compounds. DBCP deraded by autotrophic nitrifying Some pesticides, those with halogenated bacteria Nitrosomonas europaea, Nitrosolobus phenol and aniline structures, become multi-formis. complexed with soil humic material, The ammonia-oxidizing enzyme ammonia , making them more stable. ammonia monooxygenase, is capable of These coupling complexes are resistant to cometabolizing these aliphatic pesticides. acid and base extractions and thermal These aerobic dehalogenations result in the  Microbial consortiums may be necessary The first steps of degradation are for the degradation of many of these dehydrogenation and dehydrochlorination. complex compounds. The dechlorination was also achieved best Eg degradation of Dalapon (2,2- under strict anaerobic conditions using dichloropropionic acid) involved aerobic C. freundii and facultative anaerobic Pseudomonas putida and Flavobacterium bacteria included Bacillaceae species that are sp. abundant in soil. The anaerobic dehalogenation of Cometabolism may be necessary for aliphatic pesticides also available and at degradation eg in lindane degradation, pH levels of 7.5 and 8.0 resulted in the alanine, leucine, pyruvate, leucine-proline most rapid dehalogenation. mixtures, formate and glucose are necessary. These substrates act as electron donors for Chlorinated cyclic aliphatic pesticides lindane reduction. The microbial degradation of cycloalkanes varies dramatically with the type of Pentachlorophenol and DDT substitution. The degradation of the halogenated aromatic The saturated, highly chlorinated pesticides under aerobic sonditions is a compounds are extremely persistent function of the degree of chlorination. under aerobic conditions. The higher the degree of chlorination the Eg lindane (γ-hexachlorocyclohexane, γ- more resistanct they become to aerobic HCH) is degraded by fungi. The white rot degradation. fungus Phanerochaete chrysosporium Many of these compounds degraded under mineralize lindane. anaerobic conditions. The chlorinated cyclic-aliphatic pesticides Pentachlorophenol (PCP) readily degrades in eg α, β and γ isomers of hexacyclohexane the environment by both chemical and are subject to anaerobic dehalogenation biological processes. by a variety of anaerobic Mos. Bacteria and fungi are active degraders of The anarobic metabolism of lindane has PCP. been demonstrated with Clostridium Microbial species include bacteria rectum, Clostridium butyricum, DDT The Degradation of DDT requires an initial anaerobic condition for reductive dehalogenation. MOS able to degrade DDT : Pseudomonas spp., P. aeruginosa, Clostridium perfringens, Bacteroides sp., E. coli, Enterobacter aerogenes and Proteus vulgaris. Chlorinated phenylalkoates Herbicides consisitng of phenylalkanoate strucutres are widely used for broad-leaved weed control by homeowners and in agriculture and highway maintenance.  A pesticide is any substance or mixture of Pesticides can be classified by target substances intended for preventing, organism, chemical structure, and physical destroying, repelling or mitigating any pest. state. A pesticide may be a chemical substance, Pesticides can also be classed as inorganic, biological agent (such as a virus or synthetic, or biologicals bacterium), antimicrobial, disinfectant or (biopesticides),although the distinction can device used against any pest. Pests include sometimes blur. insects, plant pathogens, weeds, molluscs, Biopesticides include microbial pesticides birds, mammals, fish, nematodes ( and biochemical pesticides.Plant-derived roundworms), and microbes that destroy pesticides, or "botanicals", have been property, spread disease or are a vector for developing quickly. These include the disease or cause a nuisance. pyrethroids, rotenoids, nicotinoids, and a Although there are benefits to the use of fourth group that includes strychnine and pesticides, there are also drawbacks, such as scilliroside. potential toxicity to humans and other Many pesticides can be grouped into animals. chemical families. Prominent insecticide FAO has defined the term of pesticide as: families include organochlorines, any substance or mixture of substances organophosphates, and carbamates. intended for preventing, destroying or Organochlorine hydrocarbons (e.g. DDT) controlling any pest, including vectors of could be separated into human or animal disease, unwanted species dichlorodiphenylethanes, cyclodiene of plants or animals causing harm during or compounds, and other related compounds. otherwise interfering with the production, They operate by disrupting the processing, storage, transport or marketing of sodium/potassium balance of the nerve food, agricultural commodities, wood and fiber, forcing the nerve to transmit wood products or animal feedstuffs, or continuously. Their toxicities vary greatly, substances which may be administered to but they have been phased out because of animals for the control of insects, arachnids their persistence and potential to or other pests in or on their bodies. The term bioaccumulate. includes substances intended for use as a Organophosphate and carbamates largely  Organophosphates are quite toxic to vertebrates, and have in some cases been Uses Pesticides are used to control organisms replaced by less toxic carbamates. Thiocarbamate and dithiocarbamates are considered harmful.For example, they are subclasses of carbamates. used to kill mosquitoes that can transmit Prominent families of herbicides include potentially deadly diseases like west nile virus pheoxy and benzoic acid herbicides (e.g. 2,4-D , yellow fever, and malaria. They can also kill ), triazines (e.g. atrazine), ureas (e.g. diuron), bees, wasps or ants that can cause allergic and Chloroacetanilides (e.g. alachlor). reactions. Phenoxy compounds tend to selectively kill Insecticides can protect animals from broadleaved weeds rather than grasses. The illnesses that can be caused by parasites such phenoxy and benzoic acid herbicides function as fleas. Pesticides can prevent sickness in humans similar to plant growth hormones, and grow cells without normal cell division, crushing the that could be caused by mouldy food or plants nutrient transport system. diseased produce. Triazines interfere with photsynthesis. Herbicides can be used to clear roadside Many commonly used pesticides are not weeds, trees and brush. They can also kill included in these families, including invasive weeds that may cause environmental glyphosate. damage. Herbicides are commonly applied in ponds Algicides or algaecides for the control of algae Avicides for the control of birds and lakes to control algae and plants such as Bactericides for the control of bacteria water grasses that can interfere with Fungicides for the control of fungi and activities like swimming and fishing and cause oomycetes the water to look or smell unpleasant. Uncontrolled pests such as termites and Herbicides (e.g. glyphosate) for the control of weeds mould can damage structures such as houses. Pesticides are used in grocery stores and Insecticides (e.g. organochlorines, organophosphates, carbamates, and food storage facilities to manage rodents and pyrethroids) for the control of insects - these insects that infest food such as grain. Each can be ovicides (substances that kill eggs), use of a pesticide carries some associated larvicides (substances that kill larvae) or risk.  Proper pesticide use decreases these Symptoms include nervous excitement, associated risks to a level deemed tremors, convulsions or death. acceptable by pesticide regulatory agencies Scientists estimate that DDT and other such as the chemicals in the organophosphate class of United States Environmental Protection Agen pesticides have saved 7 million human cy lives since 1945 by preventing the (EPA) and the Pest Management Regulatory transmission of diseases such as malaria, Agency (PMRA) of Canada. Pesticides can save farmers' money by bubonic plague, sleeping sickness, and typhus. preventing crop losses to insects and other However, DDT use is not always effective, pests; in the U.S., farmers get an estimated as resistance to DDT was identified in fourfold return on money they spend on Africa as early as 1955, and by 1972 pesticides. One study found that not using pesticides nineteen species of mosquito worldwide were resistant to DDT. reduced crop yields by about 10%.Another A study for the World Health Organization study,conducted in 1999, found that a ban in 2000 from Vietnam established that on pesticides in the United States may result non-DDT malaria controls were in a rise of food prices, loss of jobs, and an significantly more effective than DDT increase in world hunger. DDT, sprayed on the walls of houses, is an use.The ecological effect of DDT on organisms is an example of organochloride that has been used to fight bioaccumulation. malaria since the 1950s. Recent policy statements by the World Health Organization have given stronger support to this approach.Dr. Arata Kochi, WHO's malaria chief, said, "One of the best tools we have against malaria is indoor residual house spraying. Of the dozen insecticides WHO has approved as safe for house spraying, the most effective is DDT.