Environmental Biotechnology PDF

Environmental Biotechnology Bioremediation Concepts, situ and ex-situ bioremediation, biodegradation of pollutants, biolixiviation, biosorption and accumulation of heavy metals, biolixiviation,… Dr. Faouzi Ben Rebah Associate Professor...

Environmental Biotechnology Bioremediation Concepts, situ and ex-situ bioremediation, biodegradation of pollutants, biolixiviation, biosorption and accumulation of heavy metals, biolixiviation,… Dr. Faouzi Ben Rebah Associate Professor Higher Institute of Biotechnology of Sfax 1 Pollution: Industrial and agricultural activities cause widespread contamination of soils and groundwater by toxic compounds. Pollution is the introduction of substances or energy into an environment that can have harmful or toxic effects on humans and the environment. Bioremediation: Bioremediation refers to all processes that eliminate organic or mineral pollutants present in natural environments through the action of microorganisms. 2  Terminology Depollution: Spontaneous or intentional elimination or inactivation of a pollutant by physical, chemical, or biological agents. Biodegradation: Partial or complete breakdown of a substance by biological agents. Biotransformation: Transformation of a substance by biological agents (incomplete metabolism). Biostimulation: Stimulation of indigenous microflora by adding nutrients and controlling physico-chemical factors (pH, temperature, humidity, oxygen). Bioaugmentation: Controlled addition of preselected microorganisms (pure strains, consortia, mixed cultures) to assist indigenous microorganisms. Bioreduction: Reduction of oxidized compounds (nitrates, metal oxides) through biological means. Biolixiviation: Extraction of metals contained in sludge, soil, sediment, or 3 mineral by solubilization induced by microorganisms. Biofixation/biosorption: Fixation of pollutants, most often metals present in a liquid effluent, onto microorganisms. Xenobiotics: Synthetic compound created by humans, not biological (foreign to the living world). Phytoremediation: Includes all technologies that use vascular plants, algae (phycroremediation), or fungi (mycoremediation) to eliminate or control contaminants or accelerate the degradation of compounds through microbial activity. Phytotransformation: Uptake of contaminants present in soil and groundwater and transformation by plants. Phytostabilization: Plants or roots keep contaminants in place, minimizing their mobility. Phytoextraction: Plants capable of accumulating contaminants in their upper parts (leaves, stems, etc.). 4  Bioremediation  Is a technology that utilizes living organisms, such as bacteria or fungi, to detoxify pollutants in soil and water through processes like biodegradation, biotransformation, and mineralization, preferably in situ.  This approach, potentially more cost-effective than traditional techniques (e.g., extraction, land spreading, electrolysis, soil incineration, water filtration with activated carbon, etc.), requires a thorough understanding of how microorganisms transform chemical compounds, how they survive in polluted environments, and how they should be applied at contaminated sites. 5  Microorganisms Source of Elctron Energy Acceptor moisture nutrients elimination absence of absence of competitive of toxicity organisms metabolites The needs of bioremediation 6  Pollutant Types Chemical compounds Metals Organic compounds  Essential to life in very  Petroleum hydrocarbons (diesel, small amounts (trace fuels, gasoline, kerosene…) elements = Na, K, Mn,  Waste from oil exploitation: Ca...), as they are involved sludge and oil residues (tar) in cellular metabolism.  Organic residues from the chemical industry: alcohols,  Non-essential to life, heavy acids… metals (Cd, Hg, Al, and  Halogenated organic Pb), generally present in compounds (herbicides, trace amounts in the fungicides, insecticides) 7 biosphere.  Bioremediation techniques  Ex situ  In situ  Bioventing  Land farming  Biosparging  Compost  Biostimulation  Biopiles  Bioaugmentation  Bioreactors  Phytoremediation  Bioventing  The most common in situ treatment supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria.  Bioventing employs low air flow rates and provides only the oxygen necessary for the biodegradation while minimizing volatilization and release of contaminants to the atmosphere.  It works for simple hydrocarbons and can be used where the contamination is deep under the surface.  Biosparging  Biosparging involves the injection of air under pressure below the water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria.  Biosparging increases the mixing in the saturated zone and there by increases the contact between soil and groundwater.  Low cost of installing small - diameter air injection points allows considerable flexibility in the design and construction of the system. Biosparging  Biostimulation  It involves supplying oxygen and nutrients by circulating aqueous solutions through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants.  It can be used for soil and groundwater.  Generally, this technique includes conditions such as the infiltration of water - containing nutrients and oxygen.  Bioaugumentation  Bioremediation frequently involves the addition of microorganisms indigenous or exogenous to the contaminated sites.  Two factors limit the use of added microbial cultures in a land treatment unit: 1) non indigenous cultures rarely compete well enough with an indigenous population to develop and sustain useful population levels 2) most soils with long-term exposure to biodegradable waste have indigenous microorganisms that effectively degrade waste, if the land treatment unit is well managed.  Land farming  It is a simple technique in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded.  The goal is to stimulate indigenous microorganisms and facilitate their aerobic degradation of contaminants.  In general, the practice is limited to the treatment of superficial 10–35 cm of soil.  Since landfarming has the potential to reduce monitoring and maintenance costs, as well as clean-up liabilities, it has received much attention as a disposal alternative.  Composting  Composting is a technique that involves combining contaminated soil with nonhazardous organic amendmants such as manure or agricultural wastes.  The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting.  Biopiles  Biopiles are a hybrid of landfarming and composting. Essentially, engineered cells are constructed as aerated composted piles.  Typically used for treatment of surface contamination with petroleum hydrocarbons they are a refined version of landfarming that tend to control physical losses of the contaminants by leaching and volatilization.  Biopiles provide a favorable environment for indigenous aerobic and anaerobic microorganisms.  Bioreactors  Slurry reactors (Slurry : mixture of solid particles suspended in a liquid, typically water.) or aqueous reactors are used for ex situ treatment of contaminated soil and water pumped up from a contaminated zone.  Bioremediation in reactors involves the processing of contaminated solid material (soil, sediment, sludge) or water within a controlled treatment system.  A slurry bioreactor can be defined as a vessel and apparatus designed to create a three-phase mixing environment (solid, liquid, and gas) that enhances the bioremediation rate of soil-bound and water-soluble pollutants. This system utilizes a water slurry of contaminated soil and biomass, typically consisting of indigenous microorganisms, that are capable of degrading the targeted contaminants.  Slurry bubble column slurry-bed reactor. Agitated slurry reactor (SBCR) reactor (ASR).  Phytoremediation  Plants have been commonly used for the bioremediation process called Phytoremedation: use of plants to decontaminate soil and water by extracting heavy metals or contaminants.  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 used to satisfy environmental regulation and costs less then other alternatives.  This process is very effective in cleaning polluted soil. Types of phytoremediation 1) Phytoextraction 2) Phytotransformation 3) Phytostabilisation 4) Phytodegradation 5) Rhizofiltration  Phytoextraction  In phytoextraction, certain plants, known as hyperaccumulators, absorb contaminants through their roots and concentrate them in their stems, leaves, or other tissues.  The plants are then harvested, and the contaminants are removed from the environment along with the plant biomass.  This method is primarily used to clean up soil contaminated with metals like lead, cadmium, zinc, or arsenic.  It offers an environmentally friendly, cost-effective alternative to traditional remediation techniques, though it can take longer and is dependent on the ability of specific plant species to accumulate large quantities of metals.  Phytotransformation  Refers to the uptake of organic contaminants from soil, sediments, or water and, subsequently, their transformation to more stable, less toxic, or less mobile form.  Chromium can be reduced from hexavalent to trivalent chromium, which is a less mobile and noncarcinogenic form.  Phytostabilization  The plants reduce the mobility and migration of contaminated soil.  Leachable constituents are adsorbed and bound into the plant structure  They form a stable mass of plant from which the contaminants will not reenter the environment.  Phytodegradation  Breakdown of contaminants through the activity existing in the rhizosphere.  This activity is due to the presence of proteins and enzymes produced by the plants or by soil organisms such as bacteria, yeast and fungi.  Rhizodegradation is a symbiotic relationship that has developed between plants and microbes.  Plants provide nutrients necessary for the microbes to grow, while microbes provide a healthier soil environment.  Rhizofiltration  It is a water remediation technique that involves the uptake of contaminants by plant roots.  Rhizofiltration is used to reduce contamination in natural wetlands and estuary areas.  Limitations of Bioremediation  Contaminant type & Concentration  Environment  Soil type condition & Proximity of ground water  Nature of organism  Cost benefit ratios : Cost Vs Env. Impact  Does not apply to all surface  Length of time of the bioremediation process  Advantages  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  Cost overrun  Failure to meet targets  Poor management  Climate Issue  Release of contaminants to environment  Unable to estimate the length of time it’s going to take, it may vary from site.  Metal Recovery A – Biolixiviation B – Biosorption C – Precipitation D - Phytoremediation 32  A- Biolixiviation  The ability of certain bacteria to extract metallic elements from minerals by solubilizing them.  Microorganisms are used to dissolve metal precipitates in order to extract metals (U, Cu, etc.).  This process is mainly used for low-grade minerals ( MSO4 35 Indirect Method : 1- Metal sulfides (MS) are oxidized purely chemically by ferric iron ions: MS + 2Fe3+ -------> M2+ + 2Fe2+ + S˚ 2- Then, the bacteria oxidize S˚, resulting in the production of H2SO4 and F2+ converted Fe3+ : S˚ + + 1,5 O2 + H2O + Thiobacilus -------> H2SO4 2Fe2+ + 0,5 O2 + 2H+ + T. ferroxydans -------> 2Fe3+ + H2O 3- Finally, from ferric iron, the cycle can restart: During this process, there is a decrease in pH and an increase in the redox potential. ===> These conditions lead to the solubilization of carbonates and metal oxides: : MO + H2SO4 -------> MSO4 + H2O MCO3 + H2SO4 -------> MSO4 + H2O + CO2 36  Biolixiviation of Gold  Solubilization by microbial oxidation at low temperature using T. ferrooxidans :  the Gold (Au⁺) is then immobilized with a cyanide (CN⁻) solution: 37  B- Biosorption  Biosorption: A general term used to describe all processes related to metal recovery in the presence of biomass.  Biosorption utilizes raw products from biomass.  The performance of sorption processes depends on the type of biomass and the chemistry of the solution.  The biosorbent can be modified to enhance its sorption capacity. 38 Main Biosorbents:  A large number of biosorbents for extracting heavy metals: Algae, Bacteria, Fungi, Yeasts, Plants  Biosorption is a property of certain types of living, inactive, or dead biomass to bind and concentrate metals.  There are two methods of extraction of metal ions by living and dead cells:  The first method: involves the binding of metal ions at the surface of the cell membrane and extracellular material: passive extraction: biosorption.  The second method: dependent on cellular metabolism: active extraction: bioaccumulation. 39 Mechanisms of Biosorption:  Biosorption is a complex phenomenon where metallic species can be deposited on the biosorbent through various sorption mechanisms: – Ion exchange – Complexation – Chelation – Precipitation – Electrostatic interactions, etc.  Ions can attach to the biomass through different physicochemical mechanisms depending on the nature of the biomass and environmental conditions: – Van der Waals forces – Covalent bonds – Redox reactions  Negatively charged groups in the biomass, such as carboxyl, hydroxyl, and phosphate groups, adsorb metallic cations. 40  Biosorption by Algae  Algae possess a large quantity of biopolymers with a high metal adsorption capacity.  There is a wide variety of algae: red, brown, and green.  Algae contain biopolymers: – Polysaccharides (cellulose, xylan, etc.) – Uronic acids (alginate acids) – Sulfated polysaccharides (agarose, agaropectin, carrageenan, etc.)  These polymers contain carboxyl, sulfonic, and hydroxyl groups.  Alginate:  Comprises 10-40% of the weight of dried algae.  Composes the cell wall and intracellular material. Contains two carboxyl groups: β-1,4 D-mannuronic acid and α-1,4 L-guluronic acid.  The affinity of alginate for cations (Pb²⁺, Cu²⁺, Zn²⁺, Ca²⁺) is significant and increases with its content of guluronic acid. 41 Biosorption by Fungi and Yeasts:  Presence of chitin.  In fungi: chitin is an essential component of the cell wall that surrounds and protects cells from the environment.  In the yeast Saccharomyces cerevisiae: chitin synthase activity. Structure of Chitin and Chitosan 42 Biosorption with Bacteria:  Active Biosorption: Metal-bacteria interactions can occur with living or metabolically active cells:  Precipitation  Intracellular accumulation  Oxidation-reduction  Methylation-demethylation  Passive Biosorption : Metals are transformed through physicochemical reactions:  Complexation by substances produced by the cells  Sorption of metal onto the bacterial cell wall: presence of active functional groups  Active and passive processes often occur simultaneously 43 Bacteria:  Bacteria have a very reactive surface.  Bacteria possess sorption sites on their cell wall.  At neutral pH, bacteria have a net negative charge.  Components of the cell wall: – Gram (+): peptidoglycan – Gram (-): outer membrane rich in groups: hydroxyls (R-OH), carboxyls (R-COOH), phosphates (R-PO₄), and amines. MP : membrane plasmique Pg : couche peptidoglycane ME: membrane extérieure 44 Factors Influencing Biosorption:  The physicochemical properties of the medium (pH, temperature, ionic strength, dissolved oxygen concentration, presence of other metallic cations, and other ligands).  Examples:  At high pH, the formation of metal hydroxides occurs, with different reactivity of cations.  At low pH, interactions between metals and organic molecules are favored.  Increased ionic strength leads to a reduction in the adsorption capacity of bacteria, regardless of the metal and the pH of the medium.  The Metal: size-to-charge ratio, ionic radius, valence, metal speciation, concentration, and solubility.  The Nature of the Biosorbent : composition and concentration. 45  C- Precipitation Metals can be precipitated as solids under oxic conditions (oxides) or anoxic conditions (sulfides).  Microbial Sulfate Reduction: The most significant process under anaerobic conditions is microbial sulfate reduction. Sulfate-reducing bacteria, such as Desulfotomaculum and Desulfovibrio, play a key role in this process. Here are some of their characteristics:  Strictly anaerobic: These bacteria thrive only in environments without oxygen.  Neutrophilic: Optimal pH is around 7.  Electron donors: They require an electron donor (e.g., lactate, formate, acetate) and some times hydrogen (H₂).  Oxidation: These bacteria perform complete or incomplete oxidation of the substrate.  Hydrogen sulfide production: Sulfate-reducing bacteria produce hydrogen sulfide, which reacts with metals to form an insoluble solid, favoring precipitation. 46 Microbial Sulfate Reduction :  Precipitation of Metals as Sulfides (Fe, Zn, Pb, Cd, …)  Metal precipitation as sulfides depends on the stability of the solid formed, with some sulfides being more stable than others (e.g., FeS is more stable than MnS).  Applications:  Groundwater treatment  Treatment of mine waters in bioreactors 47  C- Precipitation  Intracellular Accumulation: Metals are initially bound to the cell wall and then transported inside the cell.  Iron Oxidation-Reduction: These reactions alter the oxidation state of certain metals, leading to their precipitation. For example, soluble Fe(II) can be oxidized to Fe(III), which is less soluble and can precipitate as a solid. Reduction of Fe(III):  Microbial reduction of Fe(III) can contribute to the formation of Fe(II) phosphates.  Iron Oxides from Microbial Oxidation of Fe(II):  Iron oxides resulting from microbial oxidation of Fe(II) incorporate several trace elements, including heavy metals. 48  D - Phytoremediation Two Strategies Currently Developed in Phytoextraction: 1-Chelator-Assisted Phytoextraction:  Involves applying a synthetic chelator, such as ethylenediaminetetraacetic acid (EDTA), to soils, which enhances lead uptake and accumulation in the aerial parts of plants.  This is the most widely developed method. 2-Continuous Phytoextraction:  Relies on the genetic and physiological capacities of plants specialized in metal uptake, translocation, and tolerance.  The aim is to identify genes that can be used to genetically modify high- biomass-producing but sensitive plants, transforming them into hyperaccumulators. 49  Pollutant biodegradation  A wide variety of chemical compounds: organometallic (methyl-mercury, etc.); hydrocarbons; pesticides: insecticides, herbicides, fungicides, etc.  Microbial degradation of chemical compounds in the environment is an important pathway for their elimination.  The biodegradation of these compounds:  series of complex biochemical reaction  involving different microorganisms  Importance of enzymes involved in these biodegradation processes  Each stage of degradation is carried out thanks to a specific enzyme generated by various microorganisms (intracellular; extracellular)  The lack of a specific enzyme can stop the degradation process: this explains why some contaminants are still present in the environment  Biodegradation mechanisms: Identify the degradation pathways of certain compounds in nature such as carbohydrates, lignins, cellulose and hemicellulose. 50  Biodegradation:  Complete oxidation or mineralization into CO2 and H2O  Complete biodegradation: decomposition of the compounds into their basic elements  Partial biodegradation: formation of less complex intermediate compounds  Biotransformation: transformation into another more stable molecule (less toxic, etc.)  Various factors control the biodegradation 51  Biodegradation Biological Factors microrganisms:  Growth  Metabolism Environmental  Acclimatization Factors  Growth kinetics  Physical factors  Bioavailability  Chemical factors  Cometabolism  Sorption Chemical Factors  Recalcitrant  Bioavailability Structure/function  toxicity  Sorption  Bioavailability  Cometabolism  Recalcitrance  Toxicity 52  Parameters that affect bioremediation  Factors governing microbial activity:  redox potential  pH  temperature  water content  nutrients  Factors Governing the mobility Macroscopic Heterogeneities: faults, fractures,  stratification Structure Texture  Adsorption and desorption factors  clay  organic matter 53 Lack of biodegradation :  Lack of nutrients ===> Nutrients Needs  Limiting environmental conditions (pH, redox potential, temperature, pH, humidity, oxygen, etc.)  Presence of toxic substances at high concentrations (hydrogen sulphide, acid, etc.)  Compounds at low concentration  Non-bioavailable compounds Bioavailability is affected by:  Sorption  Presence in non-aqueous solution  State of the pollutant in the physical matrix and the aquifer  Complexity  Solubility 54  Recalcitrant pollutants and biodegradable pollutants A compound is qualified as recalcitrant when its biological elimination is very slow or even impossible to measure.  Examples of recalcitrant compounds: polymers (PVC, Teflon, etc.), polychlorinated organic compounds (PCB: Polychlorobiphenyl, DDT: Dichlorodiphenyltrichloroethane and PCP: pentachlorophenol)  Many xenobiotics can be degraded by microorganisms (for some this degradation can even be very rapid)  There are recalcitrant natural substances (wood lignin, humic acids, etc.)  A xenobiotic is not necessarily recalcitrant and conversely natural compounds can be recalcitrant.  Recalcitrant xenobiotic compounds are undesirable in the environment for several reasons:  Increase the risks of toxicity for sensitive species  Bioaccumulation process in the trophic chains make them potentially dangerous  Mobile recalcitrant compounds are the most dangerous: After a long period of time, they will be found in aquifers used for the production of drinking water.  Lack of knowledge of the medium and long term effects of 55 recalcitrant substances on the environment.  Recalcitrant compounds can be classified into three groups: (1) Compounds resistant to any microbial attack and which are not metabolized under any conditions (2) Compounds metabolized very slowly in nature, but which can be degraded rapidly in a dense culture of appropriate microorganisms (3) Compounds which in situ are metabolized quickly in certain environments and very slowly in others (anaerobic, aerobic conditions) 56  Six conditions must be met for a substrate to be biodegradable: 1/ An enzyme capable of carrying out the degradation 2/ The microorganism possessing the enzyme must be present in the same environment as the substrate. 3/ The molecule to be degraded should be under available state for use by microorganism (adsorbed molecules are not biodegradable) 4/ If the enzyme is intracellular, which is the case for many low molecular weight substrates, the substrate must penetrate inside the cell (the structure of the molecule and the permeability of the membrane are also involved) 5/ In the case of an inducible enzyme, the induction conditions must be met (sufficient concentration of substrate or presence of another substrate allowing cometabolism; a substrate rapidly degraded in the laboratory by cometabolism will not be degraded in situ due to lack of another substrate) 6/ The environmental conditions must be favorable for microbial growth 57  The recalcitrance of a compound is related to :  Molecule characteristics Physiological limits of living organisms  Properties of the environment where the compound is introduced  Studies of recalcitrance must take into account chemical, microbiological and environmental factors. Half-life and persistence of pollutants Time to degrade 50% of the pollutant (in hours, days and even years) The half-life time depends on:  Pollutant (persistent or degradable)  Environmental conditions (aerobiosis/anaerobiosis; intensity of microbial activity, temperature, pH, etc.) Persistence time Time to degrade 90% of the pollutant (or no detection) 58 Microbial depollution mechanisms Degradation : ===> H2O, CO2, CH4, H2, chloride, acetate…. Mineralisation ===> CO2, part of the substrate is assimilated Sometimes inhibited by some compounds: exp. glucose and amino acids inhibit the degradation of toluene, phenol, ethylene di-bromide and p-nitrophenol Cometabolisme  Unexpected conversion due to low enzyme specificity  Three possible explanations:  The substrate enters the cell in too small a quantity to activate the synthesis of enzymes capable of metabolizing it;  The substrate is degraded into unusable compounds;  Degradation of the xenobiotic involves reduction reaction and the electrons must come from a second substrate Detoxifying transformations transformations 59 Modification, sometimes minor, of the molecule which makes it inactive  Metabolism of the decomposers Nutrition of microorganisms in five phases: 1- Transport of food from the liquid to the surface of the bacteria 2- Adsorption of food on the cell membrane 3- Pre-digestion by exoenzymes or surface enzymes, to reduce the dimensions of the molecules 4- Permeation or crossing of the cell membrane. 60 5- Degradation Cometabolism of TCE by methane mono oxygenase in the presence of methane formaldehyde 61 Detoxifying transformations 1) Hydrolysis 2) Hydroxylation 3) Dehalogenation 4) Demethylation and other dealkylations 5) Methylation 6) Reduction of a NO2 function 7) Deamination 8) Cleavage of an ether bond (C-O-C) 9) Conversion of a nitrile to amide 10) Conjugation 11) Opening of benzene rings 12) Multiple reactions 13) Non-detoxifying transformation 62  1- Hydrolyse Hydrolysis of an ester bond ===> inactivates the insecticide Malathion 2- Hydroxylation Replacement of an H or OH inactivates the 2,4-D (2,4 dichlorophenoxyacetic) herbicide 3- Dehalogénation  Replacement of halogen by H ion (reductive dehalogenation): 63  Replacement of halogen by OH ion (hydrolytic dehalogenation):  Removal of halogen and an adjacent H (dehydro-dehalogenation): ===> In these reactions, the halogen is released in the inorganic form 64 4- Demethylation and other dealkylations  The dealkylation of an N- or an O- inactivates many pesticides:  Demethylation inactivates Diuron and Chloroneb  Loss of N-ethyl inactivates artrazine and triazines 65 5- Methylation  The reverse reaction (addition of a methyl group) can inactivate toxic phenols Fungicides: penta and tetra-chlorophenols; penta-cloro-nitrobenzene, pentachloroaniline 6- Reduction of NO2function pentachloroaniline 7- Deamination 66  9- Conversion of a nitrile to 8- Cleavage of an ether bond amide : (C-O-C) : 2-6- dichloro-benzonitrile (selective herbicide) ==> 2.6 – inactive dichlorobenzamide 10- Conjugation Cunninghamella elegans : Pyrene (C16H10 polycyclic aromatic hydrocarbons) + glucose ===> inactivation Dithiocarbamate (fungicide) + butyric acid ===> less toxic compound than the fungicide 67 11- Opening of benzene rings Carried out by methanogenic bacteria 12- Multiple reactions By a microorganism or a microbial concorsum (eg: Malathion) 13- Non-detoxifying transformation: transformations produce compounds: also toxic; more toxic, with new toxicity spectrum 68 Examples of dehalogenation that inactivate toxic compounds - COOH 69  Biodegradation of Hydrocarbons Hydrocarbons Alkane C-C Alkene C=C Alkyne C≡C Cycloalkanes Aromatics Complex aromatics 70  Environmental Factors Affecting Hydrocarbon Biodegradation:  Temperature: Cold environments show slower degradation rates, partly due to increased viscosity of hydrocarbons and reduced volatility. This leads to the persistence of short-chain alkanes (which are more toxic) and a decrease in enzymatic activity.  Oxygen: As an effective electron acceptor, oxygen promotes the degradation reaction. Well-aerated areas support faster degradation rates. In contrast, aquifers have limited oxygen, so microorganisms must rely on alternative electron acceptors for biodegradation.  Nutrients (N and P): Typically present in low concentrations in natural environments, especially in aquifers. Adding nutrients can enhance degradation, and a C:N. Ratio of 100:10:1 is often sufficient for hydrocarbon biodegradation.  pH: The highest degradation rates are generally observed under neutral to slightly alkaline conditions (around pH 8). In acidic conditions, fungi can degrade hydrocarbons, though at a slower rate compared to bacteria.  Water Saturation: Degradation is accelerated when water content is between 38% and 81%. At 100% water saturation, oxygen diffuses more slowly, potentially leading to anaerobic conditions, which slow down degradation. 71  Dispersion of Hydrocarbons  In aquatic environments, hydrocarbons tend to disperse at the water's surface, providing easy access for microorganisms that can degrade these compounds.  In soils and aquifers, hydrocarbons distribute among four phases: 1. Dense Non-Aqueous Liquid Phase (DNAPL): Hydrocarbons that are liquid but not water-soluble, sinking to the bottom. 2. Adsorbed Phase: Hydrocarbons attached to soil or sediment particles, which can affect their availability to microorganisms. 3. Vapor Phase: Hydrocarbons that exist as vapors in the air spaces of soil or water. 4. Dissolved Phase: Hydrocarbons that have dissolved in water, which can be more readily available for microbial degradation. The bioavailability of hydrocarbons is influenced by their solubility and specific surface area, determining how easily microorganisms can access and degrade these compounds. 72  Microorganisms Involved in Hydrocarbon Degradation Several types of microorganisms, including bacteria, yeasts, and filamentous fungi, are capable of degrading various types of hydrocarbons.  Bacteria:  Heterotrophic Bacteria: These bacteria utilize the carbon from hydrocarbons as a carbon and energy source for biomass production. Notable genera including Bacillus, Pseudomonas, Mycobacteria, Actinomycetes, especially Nocardia spp.  Autotrophic Bacteria: These bacteria metabolize sulfur compounds found in petroleum. Key examples including: Thiobacillus: Metabolizes sulfur (S) into sulfuric acid (H₂SO₄). Desulfovibrio: Under anaerobic conditions, it converts sulfur (S) into sulfide. The production of sulfides and sulfuric acid can damage metal reservoirs, making it important to control the growth of these microorganisms in practical applications.  Fungi:  Several fungal species are active in the degradation of hydrocarbons in both soil and water environments, including: Trichoderma, Aspergillus, Cladosporium. These microorganisms play a crucial role in bioremediation efforts by breaking down complex hydrocarbon compounds into less harmful substances. 73  Biodegradation rate  The degradation rate by microorganisms varies depending on the type of hydrocarbons involved, as different microorganisms exhibit distinct activity spectra. n-alkanes  Mechanisms of Action A) Hydroxylation at C1. B) Hydroperoxidation. C) Dehydrogenation reaction. D) Subterminal reactions. 74  Aerobic Degradation of Aliphatic Compounds  Alkanes: Their molecular structure is similar to that of natural compounds (long carbon chains like fatty acids, etc.), making them easily utilized as a carbon source by microorganisms. This leads to:  Incorporation of an oxygen atom onto a free carbon atom with the enzyme monooxygenase, resulting in the production of an alcohol.  Incorporation of two oxygen atoms with the enzyme dioxygenase, leading to the production of a fatty acid.  The chain length significantly affects biodegradability. The ease of biodegradability decreases based on the degree of saturation. The number and type of radicals on the aliphatic chains: radicals reduce biodegradability. 75  Aerobic Degradation of Aliphatic Compounds Halogenated Aliphatic Compounds: Chlorinated Solvents ==> Degradation is slower than that of non-chlorinated aliphatic compounds. Example: TCE (trichloroethylene, C₂Cl₄) 1.Substitution of Cl⁻ with OH⁻ 2.Oxidation catalyzed by monooxygenase and dioxygenase. 76 Note: molecular oxygen is required 77 Note: No molecular oxygen is required 78 Note that the first three processes deal with a terminal -CH3 group while subterminal oxidation process "splits" the alkane at a subterminal site. Note: molecular oxygen is required 79  Aromatic Hydrocarbons Ortho :1,2 Meta :1,3 para : 1,4  Aromatic Compounds: BTEX (Benzene, Toluene, Ethylbenzene, Xylene) and PAHs (polycyclic aromatic hydrocarbons: more than two fused aromatic rings). 80  Biodegradation involves two main steps: 1.Hydroxylation: Incorporation of a hydroxyl ion onto the aromatic ring, a reaction catalyzed by monooxygenase or dioxygenase, resulting in the formation of catechol. 2.Cleavage of the Aromatic Ring: The aromatic ring of catechol is cleaved by a second enzyme, dioxygenase, and this process continues until complete degradation occurs. Metabolism of Aromatic Compounds. The steps in the mineralization of natural aromatic compounds (aerobic degradation): 1. Hydrolysis of polymers, 2. Decarboxylation and methylation (leading to easily cleavable aromatic monomers). This process is carried out by bacteria, yeasts, fungi, and algae. The initial reactions involved in the metabolism of aromatic compounds are the hydroxylations of the aromatic ring with molecular oxygen, mediated by a group of 81 enzymes called oxygenases.  Oxygenases Oxygenases are divided into two groups: 1-The first group is known by several names: monooxygenases, hydroxylases, or mixed-function oxidases. These enzymes attach one oxygen atom to the substrates and reduce the second oxygen atom to water through the intermediate reduced enzymatic cofactor: 82  2- The second group includes oxygenases that attach two oxygen atoms to the substrate without concomitant formation of water. An example is the hydroxylation of benzoic acid to catechol: 83  In several cases, in yeasts and fungi, the initial reaction of the breaking up of the aromatic ring is the insertion of two hydroxyl groups in the ortho position relative to each other.  The further degradation of catechol or protocatechuic acid (substituted catechol) formed by the insertion of two hydroxyl groups can occur through one of two pathways known as ortho or meta cleavage.  The compounds resulting from the cleavage of various aromatic compounds are then completely oxidized to CO₂ and H₂O when utilized in the Krebs cycle. 84 Ortho cleavage results in the formation of cis,cis-muconic acid. Subsequently, it is converted into α-ketoadipic acid, which in the final step is cleaved into acetyl- CoA and succinyl-CoA. 85 -Meta cleavage occurs via a dioxygenase and forms 2-hydroxymuconic semialdehyde. In this case, the final products derived from catechol are pyruvic acid and acetaldehyde. 86  Both types of cleavage can occur within the same organism, such as in bacteria of the genus Pseudomonas, as shown in the following figure: métaclivage orthoclivage 87 General Metabolism of Aromatic Compounds 88 Aerobic Degradation of BTEX (BTEX refers to Benzene, Toluene, Ethylbenzene, and Xylenes.) 89  Benzene C6H6  The addition of another substituent has a significant effect on the biodegradation rate:  A single substituent on benzene affects the degradation order: (COOH or OH ---> NH2 ---> OCH3 ---> SO3H ---> NO2 (increases persistence).  Meta position of halogens on phenol: high persistence  Ortho and para: less effect  Increasing the number of chlorine or bromine atoms increases the molecule's persistence. 90 Case of Benzene C₆H₆: Increase in Half-Life 91  Aerobic Degradation of benzene Hydroxylation of the aromatic ring with molecular oxygen, mediated by oxygenases, leading to the formation of catechol  Cleavage of catechol, progressing to complete degradation 92 Ortho or para cleavage of catechol 93 94 Substitution of pheno Phenol Substituent Decomposition period half-life (Days) None 2 OH 2-chloro- 14 3-chloro- +72 -Cl 4-chloro- 9 -Br, ….etc. 2-bromo- 14 3-bromo- +72 4-bromo- 16 2,4-dichloro- 9 2,5-dichloro- +72 2,4,5-trichloro- +72 2,4,6-trichloro- 6 2,3,4,5-tetrachloro- +72 95 Pentachlorophenol +72 Phénol Acide parahydroxybenzoïque 96 97  Dehalogenation mechanisms of haloaromatic compounds In most cases, dehalogenation occurs after the rupture of the ring. However, direct halogenation (without breaking the aromatic ring) has been demonstrated: 3 cases of dehalogenation reactions. 1- Oxidative dehalogenation: Aerobic; R, X- R: COOH, O2 NH3 X: F, Cl dioxygénase 2- Hydroxylating dehalogenation: Aerobic; X- R: COOH, H2O NH3 X: F, Cl hydroxylase 98 3- Reductive dehalogenation: Anaerobic. X- R: COOH, H NH3 X: F, Cl halogénase 99  Polyaromatic hydrocarbons (PAHs)  PAHs: multiple associated rings.  Resistance to biodegradation ==> accumulation in the environment.  They are toxic and carcinogenic.  More than 70 compounds classified as PAHs.  They are detected in soils and sediments; a significant amount is found in industrial and domestic wastewater (causing problems in treatment systems).  Combustion of fossil fuels (coal, oil, etc.).  Natural origin: coal deposits, products synthesized by plants (terpenes, sterols, and quinones (volatilization and condensation into PAHs).  Decomposition of lignin into humic compounds ==> Production of PAHs.  Natural biological origin (some bacteria and algae). 100  Chap 5-IV Examples of PAHs : A listing of more than 650 PAHs and their chemical names, properties and CHIME molecular diagrams from NIST (Special Publication 922) 101  Factors Affecting the Degradation of PAHs: 1.Number of Rings: 1. With more than 3 rings: insignificant oxidation. 1. 4 rings: negligible oxidation rate. 2. 5 rings: insignificant oxidation. 2.Number and Position of Substituents on the Ring: 1. Example: Naphthalene with small alkyl groups (methyl, vinyl, or ethyl) is oxidized rapidly. 2. A substituent in position 2 promotes faster oxidation compared to a substituent in position 1 (less steric hindrance). 3. The substitution of naphthalene with more methyl groups on a ring reduces the oxidation rate. 3.Degree of Saturation of the Rings: 1. An increase in the number of saturated rings leads to a reduction in oxidation. 102 Insecticides : Example 1: DDT and analogues Dichlorodiphenyltrichloroethane (DDT)  The analogues are the results of DDT degradation.  The main reaction involves intermediates (DDE, etc.):  Reductive dechlorination reaction (by bacteria in soil and water).  There is less research on the metabolic products DDT of DDT. 103  Biodegradation of DDT dechlorination dechlorination 104 105  Organophosphorus Compounds Organophosphates  This group includes several insecticides (malathion, parathion, etc.).  Less persistent than organochlorine insecticides (months, weeks).  Metabolized by several bacteria (Pseudomonas, Arthrobacter, Streptomyces, and Thiobacillus) and fungi (Trichoderma). Malathion 106 Parathion Multiple attack sites involving different enzymes: Organophosphates A = phosphatase and mixed-function oxidase B = mixed-function oxidase C = phosphatase D = carboxyl esterase  Several degradation products. Malathion 107