by dr. ahmed z. abdel azeiz college of biotechnology misr university for science and technology...

35
By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: [email protected] [email protected] Tel:002- 01228188759

Upload: alexis-zimmerman

Post on 27-Mar-2015

227 views

Category:

Documents


6 download

TRANSCRIPT

Page 1: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

ByDr. Ahmed Z. Abdel Azeiz

College of BiotechnologyMisr University for Science and TechnologyE-mail: [email protected] [email protected]:002- 01228188759

Page 2: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Contents:1- Definitions.2-Bioremediation related topics.3- Stages of a biodegradation study.4- Cells and enzymes immobilization.5- Phytoremediation.6- Bioremediation of metals contaminated environment.7- Biosurfactants8- Bioremediation techniques: a) Soil bioremediation. b) Water and gasses bioremediation.

Page 3: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Bioremediation is any process that uses organisms (microorganism, algae and plant) or their enzymes to return the polluted environment to its original condition.

Biodegradation is the use of these organisms in the degradation of different pollutants.

Xenobiotic compounds are chemical compounds found in an organism but it are not normally produced or expected to be present in it.

Cometabolism: in this process the microorganism produces an enzyme to utilizes its nutrients, but by chance this enzyme can degrade a pollutant.

Page 4: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Bioremediation is a triple-corners process:

Organisms

Pollutants

Environments

Microorganisms

Plants

Enzymes

Soil

Water

Air

OrganicInorganic

Solid

Liquid

Gas

Page 5: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

BiosurfactantsBioremediation

techniques

Environments

Organisms

PhytoremediationBioremediation of

metals polluted environment

Enzymes separation and identification

Cells immobilization

Pollutants

Bioremediation

Enzymes immobilization

Page 6: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

1 -Isolation of the microorganism

5 -Determination of the biodegradation efficiency

4 -Optimization of the biodegradation conditions

3 -Identification of the microbial isolate

2 -Purification of the obtained isolates

6 -Identification of the biodegradation products.

7 -Cell or enzyme immobilization.

8 -Enzyme identification.

Page 7: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

- Isolation of bacteria and fungi is performed on Mineral Salt Medium (MSM) supplemented with the pollutant to be biodegraded as a sole source of carbon (enriched technique).

- This medium can be used as liquid or solid.

Soil pollutant ControlIsolation of petroleum biodegrading bacteria from soil and petroleum pollution.

Isolation of pesticide biodegrading bacteria from soil.

Control

Page 8: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

The medium has a pH 7 is preferred for isolation of bacteria, while pH 4.5– 5.5 for fungi.

Fig. 2: Isolation of fungi (A) and bacteria (B) from the pollutant on solid MSM.

A B

Page 9: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

This is simply performed by streaking of a lope of the MSM microbial culture or by pouring dishes technique.

The used medium is solid MSM supplemented with the pollutant as a sole source of carbon.

Purification by streakingPurification by pouring

Page 10: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

1- Spectrophotometer.2- HPLC3- GC/MS

Page 11: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

4- Using the redox indicator 2,6-dichlorophenol indophenol (DCPIP).

- The principle of this technique is that, during the microbial oxidation of the carbon source, electrons are transferred to electron acceptors such as O2, nitrates and sulphate.

- DCPIP is an electron acceptor. - The efficiency is determined by observing the

color change of DCPIP from blue (oxidized) to colorless (reduced).

Page 12: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Immobilized enzymes (or cells) is defined as the enzyme that physically or chemically confined in defined materials with retention of its catalytic activity.

The immobilization methods

1- Carrier-binding methods.

2 -Cross-linking methods.

3- Entrapping methods.

1 (Covalent binding methods

2- Ionic binding methods.

3- Physical adsorption.

4- Bio-specific binding methods.

3- Membrane.

2- Microcapsule.

1- lattice method.

4- Reversed micelle.

Page 13: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

1- Polysaccharides: cellulose, dextran and agarose derivatives.

2- Proteins: gelatin, albumin.3- Synthetic polymers: Polystyrene derivatives, ion

exchange resins, polyurethane.4- Inorganic materials: glass, sand, ceramic and

magnetite.

Page 14: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

1) Covalent binding methods include:a) Cyanogen Bromide method (CNBr).b) Acid-azide derivative methodc) Condensing reagent methodsd) Diazo coupling methodse) Alkylation methods.

Figure1: Steps of CNBr enzyme immobilization method

Page 15: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

By cross-linking of the enzyme molecules by reacting with glutaraldehyde.

Page 16: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

a) Lattice type:polyacrylamide, calcium algenate, polyvinylalcohol polymers.

b) Microcapsule type:Interfacial Polymerization Method.Liquid drying method.

Page 17: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg
Page 18: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Phytoremediation is use of plants for accumulation, removal or conversion of pollutants.

1-.

Phytoremediation

Phytostabilization

Phytotransformation Phytoextraction

Phytovolatilization Phytostimulation

Page 19: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg
Page 20: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Approximately 400 plant species have been classified as hyperaccumulators of heavy metals, such as grasses, sunflower, corn, hemp, flax, alfalfa, tobacco, willow, Indian mustard, poplar, water hyacinth, etc.

Page 21: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

The root exudates of these plants play an important role in phytoremediation as it activate the surrounded microorganisms.

Genetic engineering are used as in case of BT protein or insect pheromones producing plants to reduce the use of pesticides.

Page 22: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Metals bioremediation mechanisms

Solubilization)Bioleaching(

Complexation(Bioaccomulation)

(Biosorption)

Metal immobilization

Precipitation

- H2S producing bacteria- Siderophores.- Metal reduction.

- Exopolysaccharide.- Lipoproteins.

- Organic acids.- Siderophores.- Root exudates.

Page 23: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

pyridine-2,6-bis(thiocarboxylic acid)

SEM images of selenium-siderophore complex formed in P. stutzeri culture

filtrate.

Chemical structure of some siderophores

Page 24: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

The biosurfactants are chemical compounds characterized by hydrophobic and hydrophilic (non-polar and polar) regions in one molecule (amphipathic molecules).

Biosurfactants from bacteria, cyanobacteria, fungi and yeast are classified into:

1) Glycolipids.2) Lipopeptides.3) Phospholipids.4) Glycoproteins.5) Polymeric biosurfactants.

Biosurfactants

Page 25: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Glucolipid from Alcaligens sp.

Glucolipid from Alcalivorax sp.

Trehalose tetra ester from Arthrobacter sp.

Chemical structure of some biosurfactants

Page 26: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

1- Increase the availability of hydrophopic compounds2- Nutrient storage molecules.3- Save the microbial cells from toxic substances.4- Efflux of harmful compounds.5- Extracellular and intracellular interactions such as

quorum sensing and biofilm.

Page 27: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

The microbe may access a poorly water-soluble substrate that has been “pseudosolubilized” by the biosurfactant.

Reduce the adsorption of the non-polar pollutants to the surface of soil particles.

Page 28: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

A) Soil bioremediation:(1) In-situ (without excavation).(2) Ex-situ (with excavation).

Only ex-situ processes allow an efficient optimization of incubation parameters (biostimulation), including: pH, Aeration, Agitation, Moisteningnutrients, solvents or surfactants.

In addition to addition of microorganisms (bioaugmentation).

Page 29: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

The ex-situ technique includes:1- Bioslurry reactor.2- Biopile.3- landfarming

Bioslurry reactor.

Biopile.

Biopile.

Page 30: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

1- High density poly ethylene (HDPE)2- Sump pump to collect leachate 3- Layer of pea gravel 4- Layer of polluted soil to be treated 5- Chopped alfalfa hay to retain moisture 6- Wheels on sprinkler piping system 7- Piping frame, aluminum or PVC pipes with

frequent holes, sufficient to allow water, nutrients and bacteria to treat the land farm plot

8- Flexible leachate collection hose

9- Bypass valve that allows leachate to be circulated directly to water distribution tank, 10- Recirculation hose 11- Alken-Murray Bioactivator 2000, bioreactor unit 12- Fresh water supply hoses 13- Pumps for fresh water 14- Treated water hose 15- Water distribution tank 16- Pump for distribution tank

Page 31: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Biofiltration is a process, in which, microorganisms supported on inert materials are used to degrade organic pollutants for air, gas and water bioremediation.

Types of biofilters: 1- Bioscrubbers. 2- Biotrickling filters. 3- Slow sand or carbon filters.

Page 32: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Bioscrubber filters

Page 33: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Slow sand or carbon filters work through the formation of a gelatinous layer (or biofilm layer) on the top few millimetres of the fine sand or carbon layer.

This layer contains bacteria, fungi, protozoa, rotifera and a range of aquatic insect larvae (i.e. rotifers).

Page 34: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg

Several research funding agency fund this type of applied research.

Now each of us can start to work in a bioremediation research.

Page 35: By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.comalrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg