biodegradation Hussein sabit

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BIODEGRADATION OF CHLORPYRIFOS BY SOME BACTERIAL STRAINS ISOLATED FROM EGYPTIAN POLLUTED SOILS:

BIODEGRADATION OF CHLORPYRIFOS BY SOME BACTERIAL STRAINS ISOLATED FROM EGYPTIAN POLLUTED SOILS By Hussein Sabit , Ph.D Assistant Professor, College of Biotechnology, MUST

Overview:

Overview Introduction Background Methodology Results and Discussion Conclusion

Introduction :

Introduction Synthetic chemicals are a beneficial and necessary part of life in any modern society. Over 363,000,000 kg of pesticide products enter our environment annually. Some 37 to 56 million tons of waste generated annually is considered hazardous. Biological toxicity is only one important aspect in understanding the environmental impact of toxic xenobiotics

Slide 4:

To further understand the potential environmental concerns, we also need to know their fate in the environment. Degradation of toxic substances may result from biotic or a biotic activities. Chlorpyrifos (CP) is an organophosphorous insecticide that is widely used for pest control in agriculture and to a lesser degree for indoor use and soil applications to control termites. CP has a high soil-absorption co-efficient, but low water solubility.

Bioremediation:

Bioremediation The use of biological organisms such as microbes or plants to aid in removing hazardous substances from an area.

More Methods:

More Methods Biodegradation. Biodegradation of organic pollutants. Behavior of Oil in the environment. Biodegradation of Oil. Bioaugmentation . Biostimulation . Hydrocarbon decomposition. Aerobic dechlorination . Microbial bioremediation.

Introduction:

Introduction Extensive production and use of synthetic organic compounds for domestic, municipal, agricultural, industrial and military activities has led to a wide distribution of these compounds in the environment Contamination of soils, groundwater, sediments, surface water and air with these hazardous compounds is one of the major problems that the world is facing today Polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), pesticides and other endocrine disruptors remain the main pollutants of concern today Organic pollutants

Introduction:

Introduction Environmental pollution due to pesticides is a global consideration Herbicides consumption in 2005 in Mauritius amounted to about 57% of the average annual consumption (2141 tons) of pesticides Atrazine , hexazinone , 2,4-D and ioxynil are commonly used by the planters in Mauritius Pesticides

Biodegradation:

Biodegradation Why study? One of few fate processes where material is gone from the environment Change concentrations that are present to have effect We can play with microbial communities to get them to do some things we want

Biodegradation:

Biodegradation Three big categories- no one told bugs Rapid breakdown- days to weeks Slow breakdown- months to years Almost no breakdown- many years Chemical structure important Biodegradation requires the presence of the appropriate organism, the chemical in an available form, and the right environmental conditions for organisms to function

Bacterial Metabolism:

Aerobic Oxidation Cometabolism Anaerobic Denitrification Manganese reduction Iron reduction Sulfate reduction Methanogenesis Bacterial Metabolism

Bioavailability:

Bioavailability AQUEOUS SORBED GASEOUS NON-AQUEOUS Not accessible Accessible

Slide 13:

Biodegradation and Mineralization Biodegradation : a biological process of reducing a compound complexity. Mineralization : a degradation process of organic compounds into inorganic one .

Biodegradation and Biotransformation:

Biodegradation and Biotransformation Conversion of contaminants to mineralized (e.g. CO 2 , H 2 O, and salts) end-products via biological mechanisms. Biotransformation refers to a biological process where the end-products are not minerals (e.g., transforming TCE to DCE ). Involves the process of extracting energy from organic chemicals via oxidation of the organic chemicals.

Biodegradation:

Biodegradation Aerobic and anaerobic degradation Reduces aqueous concentrations of contaminant Reduction of contaminant mass Most significant process resulting in reduction of contaminant mass in a system

Fundamentals of Biodegradation:

Fundamentals of Biodegradation All organics are biodegradable, BUT biodegradation requires specific conditions There is no Superbug - not Volkswagon Contaminants must be bioavailable Biodegradation rate and extent is controlled by a “limiting factor”

How do they grow: requirements for biodegradation?:

How do they grow: requirements for biodegradation? Nutrients Carbon, Nitrogen, Phosphorus, Sulfur Many chemicals supply these Micronutrients/ trace metals/ vitamins Electron acceptors - usually O 2 Converts / burns carbon substrate to CO 2 Energy and biomass ie GROWTH

Slide 18:

Biodegradation SINGLE BACTERIUM 2.0 m ORGANIC POLLUTANT AND NUTRIENTS ( C,P,N,O,Fe,S ……) GROWTH - CELL DIVISION INCREASE IN BIOMASS CO 2 evolved O 2 consumption Controlled release of energy Slow Burning!

Dehalogenation:

Dehalogenation Dehalogenation refers to the process of stripping halogens (generally Chlorine) from an organic molecule Dehalogenation is generally an anaerobic process, and is often referred to as reductive dechlorination R– Cl + 2e – + H + ––> R–H + Cl – Can occur via dehalorespiration or cometabolism Some rare cases show cometabolic dechlorination in an aerobic environment

Basic Metabolism Process of Bacteria:

Basic Metabolism Process of Bacteria Growth and Reproduction Catalyzed by Enzymes CELL ENERGY SOURCE NUTRIENTS CARBON SOURCE NEW CELL MASS H 2 O CO 2

Schematic Diagram of Biodegradation:

Schematic Diagram of Biodegradation Oil Microbe CO 2 +H 2 O CO 2 +H 2 O CO 2 +H 2 O Microorganisms eat oil and other organic contaminants. Microorganisms digest oil and convert it to CO 2 and H 2 0 Microorganisms release CO 2 and H 2 0 1. 2. 3.

Sources Natural vs. Anthropogenic :

Sources Natural vs. Anthropogenic Pictures from various sources

BIOREMEDIATION & BIODEGRADATION:

BIOREMEDIATION & BIODEGRADATION

THE BASIC PROBLEM: RELEASE OF HAZARDOUS MATERIALS:

THE BASIC PROBLEM: RELEASE OF HAZARDOUS MATERIALS Enormous quantities of organic & inorganic compounds are released into the environment each year as a result of human activities. The release may be: Deliberate and well regulated (industrial emissions) Accidental and largely unavoidable (chemical/oil spills) US EPA estimated that in 1980 at least 57 millions metric tons of the total waste can be categorized into three general groups:

Slide 27:

Heavy metal, Pb , Hg, Cd , Ni and Be can accumulate in various organs, interfere with normal enzymatic reactions and cause disease including cancer Chlorinated hydrocarbons, also known as organochlorides including pesticides and other organic compounds such as PCB (polychlorinated biphenyls) Research proven a positive correlation between cancer in lab animals and organochlorides . Nuclear waste including radioactive material such as plutonium which are dangerous for thousands of years

BIOREMEDIATION:

BIOREMEDIATION Bioremediation is the application of biological process principles to the treatment of groundwater, soil and sludges contaminated with hazardous chemicals. It requires the control and manipulation of microbial processes in surface reactors or in the subsurface. The contaminants can be biodegraded in situ or removed and placed in bioreactor (at or off the contamination sites). Idea: To isolate microbes that can degrade or eat a particular contaminant To provide the conditions whereby it can do this most effectively, thereby eliminating the contaminant

BIODEGRADATION:

The breakdown of organic compounds by micro-organisms How might microorganisms attack hazardous organic wastes? Mineralize compound directly, compound converted to harmless inorganic molecules such as carbon dioxide and salts Of prime importance are microorganisms capable of producing enzymes that will degrade the hazardous chemical (target compound) as enzymes degrade compounds through exploitation of the organism’s energy need. Converting compound to some other compound, which may also be toxic and recalcitrant to further degradation BIODEGRADATION

Slide 30:

Heterotrophic microorganisms are the principal user of organic matter in the biosphere and are key in cycling carbon from the organic to the inorganic state. Provided that sufficient inorganic nutrients as an energy source and a terminal electron acceptor for metabolism are present, all naturally occurring organic material can be biodegraded eventually. CONCEPTS:

Slide 31:

Simple organic compounds such as acetate may persist under condition that do not favor microbial activity. These conditions include extremes in temperature or pH, the presence of toxicants or antimicrobial agents, the inhibition or exclusion of microbial enzymes, and the lack of water and an electron acceptor. CONCEPTS:

REQUIREMENTS FOR BIOREMEDIATION:

REQUIREMENTS FOR BIOREMEDIATION MICROORGANISMS ENERGY SOURCE ELECTRON ACCEPTOR MOISTURE pH NUTRIENTS TEMPERATURE ABSENCE OF TOXICITY REMOVAL OF METABOLITIES ABSENCE OF COMPETITIVE ORGANISMS BIOREMEDIATION

Slide 33:

Microorganisms destroy organic contaminants in the course of using the chemicals for their own growth and reproduction. Organic chemicals provide: carbon, source of cell building material, electrons, source of energy

Slide 34:

Metabolism is defined by the nature of the redox reaction Metabolism modes are divided into two; aerobic and anaerobic Cells catalyze oxidation of organic chemicals (electron donors), causing transfer of electrons from organic chemicals to some electron acceptor Electron acceptors: In aerobic oxidation, acceptor is oxygen In anaerobic, acceptor is: -nitrate -manganese -iron -sulfate

TYPES OF BIOREMEDIATION:

TYPES OF BIOREMEDIATION The two main types of bioremediation are in situ bioremediation and ex situ bioremediation. In addition, another offshoot of bioremediation is phytoremediation .

In Situ Bioremediation:

In Situ Bioremediation In situ bioremediation is when the contaminated site is cleaned up exactly where it occurred. It is the most commonly used type of bioremediation because it is the cheapest and most efficient , so it’s generally better to use. There are two main types of in situ bioremediation: intrinsic bioremediation and accelerated bioremediation.

Intrinsic Bioremediation:

Intrinsic Bioremediation Intrinsic bioremediation uses microorganisms already present in the environment to biodegrade harmful contaminant. There is no human intervention involved in this type of bioremediation, and since it is the cheapest means of bioremediation available, it is the most commonly used. When intrinsic bioremediation isn’t feasible, scientists turn next to accelerated bioremediation.

Accelerated Bioremediation:

Accelerated Bioremediation In accelerated bioremediation , either substrate or nutrients are added to the environment to help break down the toxic spill by making the microorganisms grow more rapidly. Usually the microorganisms are indigenous, but occasionally microorganisms that are very efficient at degrading a certain contaminant are additionally added.

Slide 39:

Main advantage is that site disturbance is minimized, which is particularly important when the contaminated plume has moved under permanent structures. Biggest limitation of in situ treatment has been the inability to deal effectively with metal contaminants mixed with organic compounds. The goal of in situ treatment is to manage and manipulate the subsurface environment to optimize microbial degradation.

In Situ Bioremediation:

Land treatments: Bioventing is the most common in situ treatment and involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria. In Situ Bioremediation

Slide 41:

In situ biodegradation involves supplying oxygen and nutrients by circulating aqueous solutions through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants. Bioaugmentation Bioremediation frequently involves the addition of microorganisms indigenous or exogenous to the contaminated sites .

Slide 42:

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 thereby increases the contact between soil and groundwater.

Ex Situ Bioremediation:

Ex Situ Bioremediation Another type of bioremediation is ex situ bioremediation, which is when contaminated land are taken out of the area to be cleaned up by the organisms. This type of bioremediation is generally used only when the site is threatened for some reason, usually by the spill that needs to be cleaned up. Ex situ bioremediation is only used when necessary because it’s expensive and damaging to the area, since the contaminated land is physically removed.

Ex Situ Bioremediation:

Landfarming is a simple technique in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded. Composting is a technique that involves combining contaminated soil with non-hazardous organic compounds such as agricultural wastes. The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting. Ex Situ Bioremediation

Slide 45:

Bioreactors- Slurry reactors or aqueous reactors are used for ex situ treatment of contaminated soil and water pumped up from a contaminated plume. Bioremediation in reactors involves the processing of contaminated solid material (soil, sediment, sludge) or water through an engineered containment system.

Phytoremediation:

Phytoremediation Phytoremediation is the use of plants to clean up potentially damaging spills. The plants work with soil organisms to transform contaminants, such as heavy metals and toxic organic compounds, into harmless or valuable forms.

Biodegradation :

Biodegradation Biodegradation – microbial catalyzed reduction in complexity of chemicals Involves the breakdown of organic compounds either through biotransformation into less complex metabolites or through mineralization into inorganic minerals, H 2 O, CO 2 or CH 4 . Mineralization - conversion of an organic substrate to inorganic end products

Slide 48:

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. The extent and rate of biodegradation depend on many factors including pH, temperature, oxygen, microbial population, degree of acclimation, accessibility of nutrients, chemical structure of the compound, cellular transport properties and chemical partitioning in growth medium.

BIODEGRADATION SYSTEM IN BIOREMEDIATION:

BIODEGRADATION SYSTEM IN BIOREMEDIATION MICROORGANISMS Growth Physiology Genetic competence Metabolic diversity Enzymology metabolites CONTAMINANTS Mass transfer Bioavailability Hydrophobicity Recalcitrance Structure Toxicity ENVIRONMENTAL FACTORS pH Temperature Moisture Oxygen Nutrients Soil type

Slide 50:

Microorganisms can be isolated from almost any environmental conditions. Microbes will adapt and grow at different temperatures, as well as extreme heat, desert conditions, in water, with an excess of oxygen, and in anaerobic conditions, with the presence of hazardous compounds or on any waste stream. Because of the adaptability of microbes and other biological systems, these can be used to degrade or remediate environmental hazards. Microorganisms

Slide 52:

Although the microorganisms are present in contaminated soil, they cannot necessarily be there in the numbers required for bioremediation of the site. Their growth and activity must be stimulated. Biostimulation usually involves the addition of nutrients and oxygen to help indigenous microorganisms. These nutrients are the basic building blocks of life and allow microbes to create the necessary enzymes to break down the contaminants. Nutrients

Slide 53:

Carbon is the most basic element of living forms and is needed in greater quantities than other elements. In addition to hydrogen, oxygen, and nitrogen it constitutes about 95% of the weight of cells. The nutritional requirement of carbon to nitrogen ratio is 10:1, and carbon to phosphorous is 30:1.

Limitations to biodegradation :

Limitations to biodegradation Adequate bacterial concentrations (although populations generally increase if there is food present) Electron acceptors Nutrients (e.g., nitrogen and phosphorus) Non-toxic conditions Minimum carbon source

Advantages of bioremediation :

Advantages of bioremediation Bioremediation is a natural process and is therefore perceived by the public as an acceptable waste treatment process Many compounds that are legally considered to be hazardous can be transformed to harmless products. Instead of transferring contaminants from one environmental medium to another, for example, from land to water or air, the complete destruction of target pollutants is possible.

Advantages of bioremediation :

Bioremediation can often be carried out on site, often without causing a major disruption of normal activities. Bioremediation is less expensive Advantages of bioremediation

Disadvantages of bioremediation :

Disadvantages of bioremediation Bioremediation is limited to those compounds that are biodegradable. There are some concerns that the products of biodegradation may be more persistent or toxic than the parent compound. It is difficult to extrapolate from bench and pilot-scale studies to full-scale field operations.

Disadvantages of bioremediation :

Bioremediation often takes longer than other treatment options Biological processes are often highly specific. Important site factors required for success include the presence of metabolically capable microbial populations, suitable environmental growth conditions, and appropriate levels of nutrients and contaminants. Disadvantages of bioremediation

Case Study: Oil spill Bioremediation:

As a result of the petroleum industry millions of tons of these compounds enter the oceans every year. Many hydrocarbons dissolve slowly in water . Others such as the aromatic compounds like benzene are more soluble, and these are toxic to living cells. While accidental releases may contribute to only a small percentage of the oil released into the marine environment large accidental oil spills receive much attention and evoke considerable public concern because they can result in contamination of ocean and shoreline environments. Case Study: Oil spill Bioremediation

Oil spill!!:

Oil spill!! The biggest spill ever occurred during the 1991 Persian Gulf war when about 240 million gallons spilled from oil terminals and tankers off the coast of Prince William Sound, Alaska. The Exxon Valdez accident at Bligh Reef in 1989 discharged 40 million litres….

Bioremediation to the rescue?:

Initial studies showed that the number of oil degrading microorganisms on oiled beaches in comparison with untreated controls increased by as much as 10,000 times. Oleophilic fertilizer enhanced biodegradation of oil. Bioremediation was a useful cleanup alternative that was used by Exxon on large scale. Bioremediation to the rescue?

Slide 63:

Oleophilic fertilizer proven to be an effective nutrient source for oil degrading microbial communities. The beaches are more compatible with local wildlife (less tendency for fur and feathers to become oiled).

Materials & Methods :

Materials & Methods Media used and chemicals: CP (95%purity)( Dursban ) was obtained from Dow- agroscience Co. Enrichment mineral salts (MS)medium has been used as a routine medium for isolation of different species of Dursban -utilizing microorganisms Soil samples: Different polluted soil samples were collected from different governorates in Egypt and used for isolation of CP utilizing bacteria. Reagent used: 4-aminoantipyrine reagent was used to measure the production of phenolic compounds in culture solution according to Crawford and Ronald et al., (1997).

Slide 65:

Isolation of CP-degrading bacteria by enrichment and screening: All bacterial isolates were screened based on the formation of degrading haloes described previously (Cho et al ., 2004) . Potential isolates were obtained and tested for their degrading ability of CP. Characterization of CP degraders: Five most potent CP degraders were characterized by the aid of Bergey’s Manual of Determinative Bacteriology (Holt et al ., 1994).

Slide 66:

Parameters controlling CP biodegradation: Five parameters were investigated for studying CP biodegradation on MS medium : Incubation period (1, 2, 3, 4, 5, 6 & 7 days), Different Dursban concentrations (1, 2, 4, 6, 8,10, 20,40, 60, 80 and 100 mls /l); Inoculum size (0.5, 1, 2, 4, 8, 10 ml/100ml); Incubation temperatures (25, 30, 35, 40 and 45ºC); Different carbon sources (D-Glucose, D-Fructose, D-Mannose, D-Ribose, Maltose, Arabinose , L- Rhamnose , Sucrose, Lactose, and starch.

Molecular genetic Identification: :

Molecular genetic Identification: R andom Amplified Polymorphic DNA (RAPD) Genomic DNA was extracted from studied isolates using Easy Quick DNA extraction kit ( Genomix ) according to the manufacturer's instructions. RAPD-PCR PCR reactions were conducted using sixe arbitrary 10-mer primers ( Operon Tech., Inc.) according to ( Tikoo et al. 2001 ).

Slide 68:

16S rRNA PCR-RFLP The amplification of 16s rDNA gene was performed according to (Watanabe et al. 2001) with some modifications with annealing temperature was 63 and the digestion of specific amplified band was carried out using ( EcoRI and. ALUI) separately. Statistical analysis The presence/absence RAPD and 16S rRNA PCR-RFLP data were analyzed using the SPSS-PC programs. Pair-wise comparisons between strains were used to calculate the genetic similarity values (F) derived from the Jaccard’s similarity.

Slide 69:

RAPD profile of soil bacterial isolates (CP5, CP6, CP7, CP8 and CP9) revealed from B5 primer (Group B5) and B3 primer (Group B3 ) .

Slide 70:

Figure2: Restriction analysis of a 16SrDNA gene fragment amplified by PCR after digestion with EcoR1 and ALU1

Selected references:

Selected references Atterby,H. , Smith,N.,Chudhry,Q . and Stead,D .(2002): Exploiting microbes and plants to clean up pesticide contaminated environment. Pesticide Outlook.13,9-13. Conville , P. S., S. H. Fischer, C. P. Cartwright, and F. G. Witebsky . 2000. Identification of Nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene. J. Clin . Microbiol . 38 : 158-164. Duncan, S., Barton, J. E. and O’Berin , P.,( 1993), Mycol. Res97,1075–1082 . Mullis, K. B., and F. Faloona . 1987. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol . 155 : 335-350. Nie , N.H.; C.H. Hull; J.G. Jenkins ; K. Steinbremmer and D.H. Bent (1975 ). Statistical package for the social sciences. 2nd edn . Megraw Hill,New York. Pandey,S . and Singh,D.K .(2004): Total bacterial and fungal population after chlorpyrifos and quinalphos treatments in groundnut ( Arachis hypogaea L .) soil. Chemosphere 55,197-205. Permaul , K., Pillay , D. and Pillay , B., Lett . Appl. (1996), Microbiol ., 23 , 307–311. Pooler, M. R., Ritche , D. F. and Hartung , J. S., (1996), Appl. Environ. Microbiol ., 62, 3121–3127. Racke,K.D.,Coats,J.R . and Titus,K.R .(1988): Degradation of chlorpyrifos and its hydrolysis products, 3,5,6- trichloro-2-pyridinol, in soil. J. Environ. Science and health B23,527-539. Racke,K.D.,Laskowski,D.A . and Schultz,M.r .(1990): Resistance of chlorpyrifos to enhanced biodegradation in soil. Journal of Agricultural and Food Chemistry 38,1430-1436. Wang,L.G.,Jiang,X.,Mao,Y.M.,Zhao,Z.HandBian,Y.R .(2005): Organophosphorous pesticide extraction and cleanup from soils and measurement using GC- NPD,Pedosphere 15,386-394. Welsh, J. and McClelland, M., (1990), Nucleic Acids Res., 18,7213–7218. Williams, J. G. K., Kubelik , A. R., Lival , K. J., Rafalski , J. A.and Tingey , S. V., (1990), Nucleic Acids Res., 18, 6531–6535.12.

Results :

Results Isolation and characterization of CP bacterial isolates: Five different bacterial isolates were selected on the basis of culture characteristics, production of halo zone around the bacterial colonies and growth in high concentrations of CP (100-300 mg /l). These five CP utilizing bacterial strains viz. B-CP5, B-CP6, B-CP7,B-CP8 and B-CP9 were characterized on the basis of Gram reaction, cell shape, cell arrangement, physiological and biochemical characteristics.

Slide 73:

Five bacterial isolates Pseudomonas stuzeri -B-CP5, Enterobacter aerogenes -B-CP6, Pseudomonas stuzeri -B-CP7, Pseudomonas maltophila –B-CP8 and Pseudomonas vesicularis -B-CP9).

Cont.:

Cont. Strain B-CP5, selected as the most potent CP-utilizing bacterial isolate. It was Gram negative and motile. It was positive in testes for catalase , KOH (3%), Oxidase , glucose and mannose fermentation, methyl red (MR), levan formation, citrate utilization, H2S production, production of amylase, pectinase and cellulase . spore formation, fermentation of arabinose , fructose, lactose, mannitol , rhamnose , starch, and sucrose sugars and nitrate reduction.

Cont.:

Cont. B-CP5: Data obtained showed that, when the Pseudomonas stutzeri -B-CP5 was grown on MS medium supplemented with 3 ml/l Dursban as sole source of carbon and energy proved to be capable of grown with 7 days as best incubation period, best Dursban concentration (0.1-0.35 ml/l), best temperature (30ºC), best pH (7), best nitrogen source (ammonium nitrate) under shaking conditions (100 rpm).

Slide 76:

RAPD and 16S rRNA PCR-RFLP RAPD and 16S rRNA PCR-RFLP techniques were used to construct the genetic fingerprinting and assess the genetic relationship and genetic distance between the studied isolates. An informative profile was obtained. The primers used produced multiple band profiles with a variable number and molecular weight size of amplified DNA fragments.

Slide 77:

Different polymorphic and monomorphic markers were obtained across RAPD profiles. As regards genetic relationships, data showed that, the highest genetic similarity was between CP8 and CP& (49.2%), while the genetic similarity between CP8 and CP was the lowest (23.0%).

Slide 78:

All isolates gave the same pattern after digestion with two restriction enzymes ( EcoRI and AluI ) except CP6 isolate. Two monomorphic bands were obtained (200and 500 bp ) among five bacterial isolates. Two specific bands for CP6 were identified (430and 140bp) with EcoRI and ALUI respectively.

Slide 79:

Parameters controlling for Dursban biodegradation by Pseudomonas stutzeri –B-CP5 Phenolic compounds were detected by 4-aminoantipyrine reagent at 610nm Incubation periods (days) Phenolic compounds Dursban Concen . (ml/l) Phenolic compounds Inoculum size (ml/l) Phenolic compounds Temperature (ºC) Phenolic compounds pH Phenolic compounds 1 0.024 0.1 0.256 0.1 0.199 20 0.123 5 0.0 2 0.045 0.15 0.233 0.2 0.298 25 0.199 5.5 0.0 3 0.084 0.2 0.21 0.4 0.244 30 0.229 6 0.199 4 0.122 0.25 0.188 0.5 0.20 35 0.21 6.5 0.25 5 0.157 0.3 0.132 1 0.142 40 0.12 7 0.299 6 0.190 0.35 0.122 2 0.121 45 0.11 7.5 0.312 7 0.234 0.4 0.02 2.5 0.122 50 0.11 8 0.12 8 0.20 0.45 0.01 5 0.1 8.5 0.12 9 0.14 0.5 0.01 9 0.11 10 0.04 0.6 0.01

Slide 80:

primer isolate Specific marker (M.W. bp) C5 CP6:Enterobacter aerogenes 250, 280, 320, 430, 550 and 740 B3 CP5: Pseudomonas stuzeri 550 and 680 CP6:Enterobacter aerogenes 500, 530 and 680 CP8:Pseudomonas maltophila 520, 580 and 630 C3 CP5: Pseudomonas stuzeri 280 and 680 CP6:Enterobacter aerogenes 480, 520, 620 and 740 CP9:Pseudomonas vesicularis 270 and 800 B5 CP5: Pseudomonas stuzeri 600 CP6:Enterobacter aerogenes 150, 620 and 750 A2 CP6:Enterobacter aerogenes 350, 520, 780, 850 and 870 CP8:Pseudomonas maltophila 580 CP9:Pseudomonas vesicularis 320 A3 CP6:Enterobacter aerogenes 230, 280, 630 and 680 CP8:Pseudomonas maltophila 430 CP9:Pseudomonas vesicularis 850 EcoRI CP6:Enterobacter aerogenes 430 AluI CP6:Enterobacter aerogenes 140 Specific markers for bacterial isolate across RAPD and16SrRNA PCR-RFLP analysis

Slide 81:

Primer Code Prime sequence Ann.Tem. Isolates Total Bands Amplified bands CP5 CP6 CP7 CP8 CP9 A2 5'-TGCCGAGCTG-3' 37 C 11 11 11 14 13 60 17 A3 5'-AGTCAGCCAC-3' 37 C 4 11 7 8 7 37 13 B3 5'-CATCCCCCTG-3' 37 C 12 9 13 10 12 56 15 C5 5'-TGCGCCCTTC-3' 37 C 11 7 9 9 11 47 11 C3 5'-GGGGGTCTTT-3' 37 C 8 7 6 8 6 35 10 B5 5'-TGCGCCCTTC-3' 37 C 7 10 8 7 8 40 18 Total 53 55 54 56 57 275 66 list of primers, their nucleotide sequences, annealing temperature and total number of bands

Slide 82:

CP5 CP6 CP7 CP8 CP9 CP5 25.3 28.8 36.6 37.0 CP6 25.3 29.4 22.9 25.3 CP7 28.8 29.4 49.2 38.2 CP8 36.6 23.0 49.1 44.8 CP9 37.0 25.3 38.2 44.8 Genetic similarity and distance values calculated from Random Amplified Polymorphic DNA bands

Dendrogram demonstrating the relationships among the five bacterial isolates :

Dendrogram demonstrating the relationships among the five bacterial isolates

Thank You for Paying Attention:

Thank You for Paying Attention

Biodegradation Biodegradation is nature's way of recycling wastes, or breaking down organic matter into nutrients that can be used by other organisms. "Degradation" means decay, and the "bio-" prefix means that the decay is carried out by a huge assortment of bacteria, fungi, insects, worms, and other organisms that eat dead material and recycle it into new forms. In nature, there is no waste because everything gets recycled. The waste products from one organism become the food for others, providing nutrients and energy while breaking down the waste organic matter. Some organic materials will break down much faster than others, but all will eventually decay. :

Biodegradation Biodegradation is nature's way of recycling wastes, or breaking down organic matter into nutrients that can be used by other organisms. "Degradation" means decay, and the "bio-" prefix means that the decay is carried out by a huge assortment of bacteria, fungi, insects, worms, and other organisms that eat dead material and recycle it into new forms. In nature, there is no waste because everything gets recycled. The waste products from one organism become the food for others, providing nutrients and energy while breaking down the waste organic matter. Some organic materials will break down much faster than others, but all will eventually decay.

Phytoremediation:

Description Use of plants to remove, destroy or sequester contaminants Applicable to wide range of media and contaminants Hydraulic control and remediation Mainly poplars for chlorinated solvents in ground water Grasses for fuels, metals in soil Contaminants Treated VOCs SVOCs Fuels Explosives Inorganics Phytoremediation http://clu-in.org/techfocus/

Phytoremediation:

Pros In situ, permanent solution Low capital and operating costs Low maintenance High hydraulic pumping pressures Reduced volume for disposal Treats wide variety of contaminants Phytoremediation Cons Shallow, low- to moderate-level contamination Lack of performance data Treatment duration Seasonally, climatically dependent Not applicable to all mixed wastes Need to displace existing facilities http://clu-in.org/techfocus/

Slide 88:

Bioremediation is defined as the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state, or to levels below concentration limits established by regulatory authorities. Wastewater, Pesticides, herbicides, petrochemicals and products ( eg . Oil, Plastics,, trichloroethene , polychlorobiphenyls ( PCB s), dioxins & dibenzofurans , and heavy metals can all persist in the environment….

Bioremediation methods:

Bioremediation methods • Use the indigenous microbial population. • Encourage the indigenous population. • Bioaugmentation; the addition of adapted or designed inoculants. • Use of genetically modified micro-organisms. • Phytoremediation.

3. Bioremediation Strategies (1/2):

90 /16 In situ bioremediation Bioventing Biosparging. Bioaugmentation. Ex situ bioremediation Land farming Composting Biopiles Bioreactors. 3. Bioremediation Strategies (1/2)

4. Advantages and Disadvantages (1/2):

91 /16 Advantages of bioremediation Bioremediation is a natural process and is therefore perceived by the public B ioremediation is useful for the complete destruction of a wide variety of contaminants. Instead of transferring contaminants from one environmental medium to another, for example, from land to water or air, the complete destruction of target pollutants is possible. Bioremediation can often be carried out on site, often without causing a major disruption of normal activities. Bioremediation can prove less expensive than other technologies that are used for cleanup of hazardous waste. 4. Advantages and Disad vantages (1/2)

4. Advantages and Disadvantages (2/2):

92 /16 4. Advantages and Disad vantages (2/2) Disadvantages of bioremediation Bioremediation is limited to those compounds that are biodegradable. Not all compounds are sus ­ ceptible to rapid and complete degradation. There are some concerns that the products of biodegradation may be more persistent or toxic than the parent compound. Biological processes are often highly specific. microbial populations, suitable environmental growth conditions, and appropriate levels of nutrients and contaminants. It is difficult to extrapolate (deduce) from bench and pilot-scale studies to fullscale field operations. Bio r e mediation often takes longer than other treat ment options.