Bioremediation methods and applications By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr University for Science and Technology E-mail: alrahman3@hotmail.com ahmed.abdelaziz@must.edu.eg Tel:002- 01228188759

Contents: 1- Definitions. 2-Bioremediation related topics 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- Biosurfactants 8- Bioremediation techniques: a) Soil bioremediation. b) Water and gasses bioremediation.

Definitions 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.

Bioremediation is a triple-corners process: Solid Inorganic Organic Liquid Organisms Pollutants Environments Gas Microorganisms Soil Water Plants Air Enzymes

Bioremediation related topics Environments Organisms Pollutants Bioremediation of metals polluted environment Phytoremediation Bioremediation Enzymes immobilization Cells immobilization Bioremediation techniques Biosurfactants Enzymes separation and identification

Stages of a biodegradation study 1- Isolation of the microorganism 2- Purification of the obtained isolates 3- Identification of the microbial isolate 4- Optimization of the biodegradation conditions 5- Determination of the biodegradation efficiency 6- Identification of the biodegradation products. 7- Cell or enzyme immobilization. 8- Enzyme identification.

1- Isolation of the microorganism - 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. Control Soil pollutant Control Isolation of petroleum biodegrading bacteria from soil and petroleum pollution. Isolation of pesticide biodegrading bacteria from soil.

B A Fig. 2: Isolation of fungi (A) and bacteria (B) from the pollutant on solid MSM. The medium has a pH 7 is preferred for isolation of bacteria, while pH 4.5– 5.5 for fungi.

2- Purification of the obtained isolate 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 pouring Purification by streaking

3- Determination of biodegradation efficiency and products: 1- Spectrophotometer. 2- HPLC 3- GC/MS

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).

Cells and Enzymes immobilization: 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 2- Cross-linking methods. 3- Entrapping methods. 1- Carrier-binding methods. 1) Covalent binding methods 1- lattice method. 2- Microcapsule. 2- Ionic binding methods. 3- Membrane. 3- Physical adsorption. 4- Bio-specific binding methods. 4- Reversed micelle.

Materials used in the Carrier-binding methods 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.

1) Covalent binding methods include: a) Cyanogen Bromide method (CNBr). b) Acid-azide derivative method c) Condensing reagent methods d) Diazo coupling methods e) Alkylation methods. Figure1: Steps of CNBr enzyme immobilization method

B- Cross-linking methods: By cross-linking of the enzyme molecules by reacting with glutaraldehyde.

3- Materials used in the entrapping methods a) Lattice type: polyacrylamide, calcium algenate, polyvinylalcohol polymers. b) Microcapsule type: Interfacial Polymerization Method. Liquid drying method.

Phytoremediation Phytoremediation is use of plants for accumulation, removal or conversion of pollutants. 1-. Phytoremediation Phytostimulation Phytostabilization Phytovolatilization Phytotransformation Phytoextraction

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.

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.

Metals bioremediation mechanisms Solubilization (Bioleaching) Complexation (Bioaccomulation) (Biosorption) Metal immobilization Precipitation - H2S producing bacteria - Siderophores. - Metal reduction. - Exopolysaccharide. - Lipoproteins. - Organic acids. - Root exudates.

Chemical structure of some siderophores pyridine-2,6-bis(thiocarboxylic acid) SEM images of selenium-siderophore complex formed in P. stutzeri culture filtrate.

Biosurfactants 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: Glycolipids. Lipopeptides. Phospholipids. Glycoproteins. Polymeric biosurfactants.

Trehalose tetra ester from Arthrobacter sp. Chemical structure of some biosurfactants Glucolipid from Alcalivorax sp. Trehalose tetra ester from Arthrobacter sp. Glucolipid from Alcaligens sp.

Physiological roles of biosurfactants: 1- Increase the availability of hydrophopic compounds 2- 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.

Biosurfactant applications in bioremediation: 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.

Bioremediation techniques: 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, Moistening nutrients, solvents or surfactants. In addition to addition of microorganisms (bioaugmentation).

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

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

B- Water and gas bioremediation: 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.

Bioscrubber filters

Slow sand or carbon filters 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).

Several research funding agency fund this type of applied research. Now each of us can start to work in a bioremediation research.

Thank you